The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typically low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules. A molecular counting approach using single molecule photobleaching reveals that the Tat receptor complex is a tetramer in its native membrane environment with an equal stoichiometry of TatB and TatC. It accomplishes this without requiring any nucleoside triphosphates (NTPs) and with the unique requirement that a proton motive force (pmf) is essential for transporting all substrates8. Despite the seemingly complex physical problem of conveying a large object across a membrane while maintaining the permeability barrier, a minimal Tat system requires only two membrane proteins, TatA and TatC, e.g., as found in some gram-positive bacteria and archaea10. Nonetheless, the best-studied Tat systems are found in Escherichia coli and in plant thylakoids, both of which additionally contain TatB, a TatA-like membrane protein. Though E. coli also contains a fourth protein, TatE, which is homologous to TatA and TatB12, E. coli TatABC comprise a common minimal system17. An early step in the transport cycle is recruitment of substrates with a twin-arginine signal peptide (containing an (S/T)RRXFLK consensus motif) by the Tat receptor complex18. However, transport cannot occur without a TatA-rich assembly, typically described as resulting from the recruitment of TatA to the receptor-substrate complex21. This active translocon is proposed to have a variable composition, allowing it to accommodate a range of mature domain sizes and shapes24. In contrast, the receptor complex is generally assumed to have a fixed composition, yet it has been surprisingly difficult to establish its oligomerization state30.The Tat machinery has the unusual capacity to transport folded proteins across energetic membranes without collapsing ion gradients32. Membranes containing TatB and TatC are sufficient for recruiting Tat substrates, establishing that these proteins form the core of the receptor complex34, and these are generally agreed to be present in a 1:1 ratio36. Whether TatA is a normal constituent of the receptor complex has been controversial; at present, the weight of evidence leans toward some TatA in the receptor complex30. More recently, it was discovered that TatA, TatB, and TatE occupy very similar positions on TatC37, suggesting that TatE is also part of the receptor complex, or that TatA takes the place of TatE when the latter is absent. Thus far, there is no evidence that any additional TatA and/or TatE within the receptor complex is in excess of one molecule per TatBC heterodimer. Thus, their presence would be insufficient to substantially increase the size of the receptor complex, although they could influence oligomerization state estimates. Due to the longstanding uncertainty regarding the presence of TatA and TatE in the receptor complex, its oligomerization state is typically discussed in terms of the number of TatBC heterodimers. Thus, the possible oligomerization states discussed throughout this paper  refer to the oligomerization state of the TatBC heterodimer within the Tat receptor complex.TatC has six transmembrane helices that generate a glove-like groove within the membrane and forms the bulk of the signal peptide binding pocket16, a fundamental structural issue is whether the signal peptide binding site is exposed to the membrane lipid interior, which it must be for a monomeric receptor complex, or whether it could be protected within the interior of a higher-order receptor oligomer38. For lipid-exposed binding site models, recruitment of TatA could, in principle, generate a translocation conduit with minimal (if any) conformational changes within the receptor complex. In contrast, for interior binding site models, substantial conformational changes appear necessary to rearrange to create a channel de novo, or to gate the channel open and closed, possibly also increasing the diameter of resting, but blocked, pore40. Notably, higher-order oligomers could form larger ‘resting’ pores, although they present a more substantial challenge to seal against ion leaks.The receptor complex oligomerization state is fundamental to a mechanistic understanding of the transport mechanism. Considering that the receptor-bound signal peptide is inserted about halfway across the membrane36. Large ~440–700 kDa complexes obtained upon detergent extraction41 can overestimate oligomer size due to the lipid and detergent content of the analyzed micelles. Crosslinking approaches28 can either under- or over-estimate complex size due to insufficient crosslinks or crosslinks to unrelated proteins. Co-evolution analysis requires inferring the correct structural model and multiple oligomeric forms may be equally viable28. While conflicting conclusions over oligomerization state can certainly point toward variable compositions as conditions change, we considered that direct measurement by molecular counting in the native membrane environment would provide a more robust result.The receptor complex oligomerization state gleaned from various studies includes a dimer, trimer, tetramer, heptamer, and octamer, although trimer and tetramer models are favored more recentlyHere, we optimized the overproduction level such that at most a few Tat receptor complexes are recovered per inverted membrane vesicle (IMV). Assuming that the cell resuspension and IMV formation processes do not disrupt the Tat receptor oligomer, the receptor complex oligomerization state in its native lipid environment can be inferred from the distribution of the Tat proteins in IMVs. Using a molecular counting approach, we show that in IMVs from cells overproducing TatABC, both TatB and TatC are present in four copies per receptor complex, the number of receptor complexes per IMV is ~1, and a large fraction of IMVs are devoid of Tat receptor complexes.7. Thus, we expected that protein complexes present within the original cytoplasmic membrane would stochastically partition into the IMVs. Once the complexes were distributed in the IMVs, our counting assay does not rely on the protein assemblies remaining intact, merely that the individual polypeptides are expected to remain associated with the same IMV.An inherent underlying assumption when purifying membrane proteins with detergents is that the extraction process does not influence the composition of the protein complex. Verification that this indeed so requires comparison with numerous controls. We avoided this complication by assaying the proteins present within native membrane vesicles. The formation of IMVs involves breaking the cell membrane into numerous pieces, which individually reseal to produce a membrane bilayer separating two aqueous compartments that in most cases is topologically inverted (the cytoplasmic leaflet is now on the outside)E. coli Tat receptor complex. We reasoned that by generating a low average number of receptor complexes per IMV via an appropriate overproduction level, the number of TatB or TatC molecules could be counted by single-molecule photobleaching methods on individually assayed IMVs. Our primary analytical assumption was that histogram distributions summarizing the number of observed steps in photobleaching traces were expected to be dependent on the oligomer size. For example, a tetramer can be expected to give rise to spots with a majority of fluorescent intensities corresponding to four, eight, twelve, etc. molecules. Thus, we expected to be able to determine for the first time the average number and distribution of TatB and TatC molecules and Tat receptor oligomers per IMV. To achieve this, we separately C-terminally tagged TatB and TatC with the fluorescent protein mNeonGreen (mNeon)42 46. Not surprisingly, then, these IMVs containing TatBmNeon or TatCmNeon exhibited reduced transport efficiencies compared with those obtained after 4 h induction ion Fig. a, b. No atB Fig. a, b; (2)eon Fig. ; and (3)47. The mNeon and CellMask channels allowed independent identification of spots containing the labeled Tat protein and CellMask-positive entities, respectively . We emphasize that the Poisson mean refers to the mean of the distribution of the number of Tat receptor complexes within the IMVs, not the mean number of photobleaching steps. For both TatBlex Fig. . This colex Fig. , and forlex Fig. .Fig. 8ThE. coli. Not surprising, then, the FDE for mNeon was somewhat lower when it was fused to the Tat proteins , indicating both that the attached Tat proteins affected fluorophore maturation, and that the FDE must be directly estimated from the experiment rather than from simple control proteins. We expect that both total molecular weight, as well as polypeptide composition, influence the fluorophore maturation of fluorescent proteins  and (iii) were addressed by including the FDE and complex distribution (Poisson mean) in the data analysis routine as fitting parameters. The FDE was dependent on the fusion construct, indicating differences in chromophore maturation efficiency for distinct fusion proteins. Background components (point iv) were eliminated by spot selection  or subtraction . Our analysis revealed that the Tat receptor complex including four copies of both TatB and TatC is the most parsimonious interpretation of the photobleaching data. We first expand on the support for this conclusion and then discuss the implications.The reported work probed the oligomerization state of the Tat receptor complex in a native membrane environment by counting photobleaching steps. While conceptually straightforward, this approach typically encounters significant challenges: (i) the photobleaching steps must be distinguishable; (ii) the interpretation relies on the FDE (which can vary depending on the fusion protein); (iii) the number of expected steps is dependent on the number of complexes in an analyzed spot; and (iv) potential interference from background components must be accounted for. Point (i) was addressed by using mNeon, a bright, relatively photostable fluorescent proteinmNeonC and three containing TatABCmNeon—yielded the same conclusion  the pentamer model (as well as other odd oligomerization states).Our conclusion that the Tat receptor complex is tetrameric with respect to both TatB and TatC is primarily based on the finding that the root-mean-square deviation (RMSD) between the experimental data and the best fit for all data sets is lowest for this oligomerization model. Six distinct preparations of IMVs—three containing TatABely Fig. . A funct; Supplementary Fig. 60. IMVs were constructed from spheroplasts, however, so it is the distribution within spheroplasts, not within intact cells, that dictates the partitioning into the resultant IMVs. We found that both TatB and TatC are fairly homogeneously distributed within spheroplast membranes  of the IMVs used for photobleaching studies Fig.  suggest 62. In such a mechanism, cargo translocation would either occur through the center of the receptor complex, or subsequent to a substantial conformational change wherein the necessary conduit is generated in a more external location. In the former case, expansion of the resting pore would be necessary, likely upon recruitment of TatA, as a tetrameric receptor complex is not expected to be able to accommodate all folded protein cargos. In either case, it is unclear if a channel exists in the center of the tetrameric receptor complex, which, if it exists, would necessarily need to be gated closed to prevent collapse of the pmf. Establishing that the Tat receptor complex contains four copies of both TatB and TatC establishes a critical framework for addressing these structural and functional issues.The mechanism of Tat translocation remains under debate. Nonetheless, most recent models place the signal peptide binding site on the inside of the Tat receptor complexE. coli strains MC4100∆TatABCDE (DADE), JM109, and BL21(λDE3) were described earlier65. Since MC4100 is an araD mutant strain whose growth is known to be inhibited by L-arabinose, an arabinose-resistant DADE strain was generated as described earlier44 and used for Tat overproduction. JM109 was used for clone amplification and plasmid maintenance. BL21(λDE3) cultures producing pre-SufI and the mNeon fluorescent standards from the indicated plasmids were grown in Luria-Bertani (LB) medium66 at 37 °C. Tat proteins were expressed in the DADE strain and grown in a modified low-salt LB media with glycerol . All cultures were supplemented with the appropriate antibiotic (30 µg/mL kanamycin or 50 µg/mL ampicillin). All new plasmids were submitted to Addgene, and their construction is described in the history of the linked SnapGene files. Coding sequences were verified by DNA sequencing. Plasmid constructions are briefly outlined below, and the encoded amino acid sequences are indicated in Supplementary Fig. The The pET-28a_mScarlet plasmid was created by amplifying mScarlet from pCytERM mScarlet N1 (Addgene #85066) with an N-terminal 6xHis-tag and an SpeI site between the 6xHis-tag and mScarlet and inserting the H6-SpeI-mScarlet PCR product into pET28a (Novagen #69864) using NcoI and BamHI. The mNeon coding sequence amplified from plasmid L40C-CRISPR.EFS.mNeon (Addgene #69146) was then used to replace mScarlet using SpeI and BamHI, generating plasmid pH6-mNeon.The mNeon coding sequence was amplified from pH6-mNeon using primers that introduced a C-terminal cysteine residue, and the PCR product was then re-inserted back into the same plasmid using SpeI and BamHI, generating plasmid pH6-mNeonC.The mNeon coding sequence was amplified from the pH6-mNeon plasmid with an SpeI site at both ends, and the PCR product was inserted upstream of the existing mNeon gene in the pH6-mNeon plasmid using SpeI, generating plasmid pH6-2xmNeon (correctly oriented product).The plasmid pH6-2xmNeonC was created identically as plasmid pH6-2xmNeon except that the PCR product containing the second mNeon gene was inserted upstream of the existing mNeon copy in the pH6-mNeonC plasmid using SpeI.mCherryC17 using KpnI and SpeI, generating plasmid pTatABmNeonC.The mNeon coding sequence was amplified from the pH6-mNeon plasmid, and the PCR product was used to replace mCherry in pBAD_TatAB67 was modified to contain restriction sites after each Tat gene using the QuikChange protocol. The new plasmid (pTatAkBxCs) contained a KpnI restriction site after TatA, an XbaI site after TatB, and a SalI site after TatC. The mNeon coding sequence was amplified from the pH6-mNeon plasmid with SalI and SphI sites at the two ends, and the PCR product was inserted downstream of TatC in pTatAkBxCs using SalI and SphI, generating plasmid pTatABCmNeon (correctly oriented product).The pTatABC plasmid600 ≈ 0.6 with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and growth was continued for 3 h at 37 °C. Cell pellets were stored at −80 °C until purification. After frozen cells were thawed on ice, they were rapidly resuspended by adding 40 mL lysis buffer  containing 1× CelLytic B , protease inhibitors , 20 µg/mL DNase, and 10 µg/mL RNase. The cell suspension was cleared by centrifugation at 40,000 × g for 20 min at 4 °C. The supernatant was loaded onto a 10 × 1 cm column with 2 mL Ni-NTA Superflow resin  that was pre-equilibrated with Buffer A . The resin was washed with 30 mL of Buffer A, 30 mL of 10 mM Tris-HCl, 1 M NaCl, pH 8, 10 mL of 10 mM Tris, 0.1 M NaCl, pH 8, and 5 mL of 10 mM Tris-HCl, 50 mM NaCl, 50% glycerol, pH 8. The bound protein was eluted with 500 mM imidazole, 50 mM NaCl, 50% glycerol, pH 8. Eluates were analyzed by SDS-PAGE to check for purity and concentration. For disulfide dimers of mNeonC and 2xmNeonC , the mNeon-containing elution fractions were incubated overnight at 4 °C and then purified using size exclusion chromatography with FPLC buffer  the next day . The mNeon genetic dimer (2xmNeon) was also purified via size exclusion chromatography using FPLC buffer (Bio-Rad NGC system with Enrich SEC 70 column).Monomeric and dimeric mNeon were obtained from 500 mL cultures inoculated with 5 mL starter cultures grown for 16 h. Protein production was induced at OD600 ≈ 0.6 with 1 mM IPTG, and 25 mL 0.5 M CAPS, pH 10 was added to raise the culture pH16. After growth for 2 h at 37 °C, cells were recovered  and then stored at -80 °C until purification. The pre-SufI(IAC) protein was purified via Ni-NTA chromatography following the same procedure used for the mNeon protein standards. To label with a fluorescent dye, purified pre-SufI(IAC) was first incubated with 1 mM tris[2‐carboxyethylphosphine] hydrochloride for 10 min, and then with a 50-fold molar excess of Alexa647 maleimide at room temperature (RT) in the dark. After 15 min, the reaction was quenched with 10 mM β-mercaptoethanol (βME). The mixture was diluted 10-fold to reduce the imidazole concentration and then re-purified by Ni-NTA chromatography.The Tat substrate pre-SufI(IAC) was overproduced from plasmid p-pre-SufI(IAC) (Addgene #168516). A starter culture (5 mL) grown for 16 h was transferred to a 500 mL culture. Protein production was induced at ODmNeonC or pTatABCmNeon. A starter culture (5 mL) grown overnight at 37 °C was used to inoculate 3 L (6 × 500 mL) of modified low-salt LB media (described earlier). Tat protein production was induced at OD600 ≈ 0.6 with 0.7% arabinose for 1 h or 4 h at 37 °C. In some cases, a 2 h “chase“ was performed by spinning down the cells after 1 h of Tat protein production, resuspending the cells in fresh media, and incubating at 37 °C for 2 h. Cells were recovered  and then stored at −80 °C until the next day. IMVs were isolated as described17, except that: (i) lysozyme was used at 1 mg/mL when making spheroplasts; (ii) the EDTA/lysozyme incubation time to make spheroplasts was 30 min; (iii) the French press was set to 16,000 psi for making IMVs; and (iv) the three step sucrose gradient used for IMV isolation was 2.1 M, 1.5 M, and 0.5 M.IMVs were prepared from the DADE strain transformed with pTatABmNeonC or pTatABCmNeon that were used to make IMVs. As described earlier7 and indicated in the previous section, spheroplasts were made before cell rupture by French press to make IMVs. To image such spheroplasts, a starter culture of modified low-salt LB media (5 mL) was grown overnight at 37 °C, and then was used to inoculate a fresh 5 mL culture in the morning. The new culture was incubated at 37 °C until OD600 ≈ 0.6, whereupon protein overproduction was initiated with 0.7% arabinose, and the induced culture was incubated for 1 h at 37 °C. Spheroplasts were made from the recovered cells . The spheroplast mixture was diluted ~1:5000 with the buffer used to make spheroplasts7, thus diluting out the EDTA and lysozyme. The diluted spheroplasts were added to a coverslip flow chamber and imaged under the microscope.Spheroplasts were prepared using the same DADE strains transformed with pTatABAlexa647 into IMVs was assayed essentially as described previously45. In short, transport reactions (35 µL) included IMVs (OD280 = 5.0) and pre-SufI(IAC)Alexa647 (50 nM) in Transport Buffer  with 1 µg/µL of BSA. Transport of substrate into the IMVs was initiated by the addition of 4 mM NADH, and continued for 20 min at 37 °C. Untransported  substrate was digested by incubating with 0.57 µg/µL proteinase K for 30 min at RT. Digestions were quenched by the addition of PMSF (5 mM) and SDS Sample Buffer . Samples were heated at 95 °C for 10 min and then analyzed on an 12% SDS-PAGE gel.Transport of pre-SufI(IAC)67 (1:2500 dilution), and polyclonal goat anti-rabbit IgG-HRP secondary antibodies .Western blots were performed using PVDF membranes, with a transfer time of 110 min at 400 mA. The transfer tank was chilled by placing it in a bucket of ice during the transfer process. TatB and TatC were detected using anti-TatB and anti-TatC primary antibodiesε650 = 270,000 cm−1 M−1 and ε506 = 116,000 cm−1 M−142, respectively. Western blot bands were visualized by chemiluminescence using the Clarity Max Western blotting kit  and the ChemiDoc imaging system. IMV concentrations were determined as the A280 in 2% SDS7.Concentrations of purified proteins were determined by the densitometry of bands on Coomassie Blue R-250 stained SDS-PAGE gels using carbonic anhydrase as a standard and a ChemiDoc MP imaging system (Bio-Rad Laboratories). Fluorescent proteins were detected by direct in‐gel fluorescence imaging using the same ChemiDoc imaging system. Alexa647 and mNeon concentrations were determined using DLS experiments were performed on Zetasizer Nano . IMV samples were diluted 1:100 with 50 mM Tris, 150 mM NaCl, pH 7.5 for analysis.Coverslips  were cleaned by sonicating in distilled water for 10 min, followed by sonicating in acetone for 10 min. After air-drying in a hood, the coverslips were plasma cleaned in argon for 5 min on the high setting . The plasma-cleaned slides were mounted in home-machined aluminum microscope holders and held in place by high-vacuum grease . Flow chambers were constructed from strips of double-sided tape and a top coverslip .47 with 488 and 640 nm diode lasers (Coherent). Images were magnified (×1.5 tube lens) and recorded using an EMCCD  or CMOS  camera. The focus was adjusted, focus-lock engaged, and the coverslip translated such that the recordings for the samples analyzed captured the photobleaching profile from the moment samples were first illuminated. This insured that no photobleaching events were missed. Exposures were 100 ms with a recording length of 500–600 frames.Single particle photobleaching analyses were performed with a Nikon Eclipse Ti using a ×100 oil-immersion objective  and narrow-field epifluorescence excitation600 = 5.0) containing TatBmNeon and TatCmNeon were first incubated with 0.1× CellMask Deep Red Plasma Membrane Stain  for 15 min at RT. The IMV solutions were then diluted with Dilution Buffer, incubated in a flow chamber for a minimum of 5 min, washed with two lane volumes of Dilution Buffer, and imaged. The same protocol was followed for staining IMV preparations with 100 pM BODIPY FL C16  and 2× FluoVolt .All buffers that were used in the single particle imaging protocol were passed through a 0.22 µm syringe filter, which substantially reduced the number of fluorescent background particles. The purified mNeon standards were diluted with Dilution Buffer , incubated in flow chambers for a minimum of 5 min, washed with two-lane volumes of Dilution Buffer (~20 µL), and then imaged. For CellMask staining, IMVs . For every 10 × 10 pixel ROI chosen, an adjacent area of the same size with no fluorescence was chosen for background subtraction. Intensities were obtained via the multi-measure function of ROI Manager. For visual step counting, photobleaching profiles were plotted using MATLAB.Fluorescent spots in photobleaching videos were selected manually using ROI Manager in the Fiji package of ImageJ48, the intensity values from photobleaching traces were used to generate pairwise difference curves using a custom MATLAB script. The pairwise differences for each trace were then binned, normalized, and plotted as a frequency histogram. The position of the last peak (highest photon level) from the histogram was used to estimate the number of photobleaching steps in the corresponding trace, as described in Supplementary Fig. For PDD analysisN measurements  using Microsoft Excel with the RiskAMP Monte Carlo plugin (Structured Data LLC) (Supplementary Software File Step expectation values were calculated assuming that Tat receptor complexes partitioned into IMVs following a Poisson distribution and the active fluorophores within receptor complexes and mNeon dimer and tetramer standards followed a binomial distribution, where the FDE defines the probability that the fluorophore is actively fluorescent (Supplementary Software Files mNeon and TatCmNeon. As discussed and indicated within the figures, the data were fit to various oligomerization models, and the best model was determined by the lowest RMSD.The main conclusions of this paper summarized in Fig. Further information on research design is available in the Peer Review FileSupplemental InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Software 1–4Reporting SummaryArticle File


Periodic strain variations are rationalised in terms of O2 adsorption or desorption during O2 exposure or CO oxidation under reducing conditions, respectively. During stoichiometric CO oxidation, the strain evolution is, however, no longer facet dependent. Large strain variations are observed in localised areas, in particular in the vicinity of the substrate/particle interface, suggesting a significant influence of the substrate on the reactivity. These findings will improve the understanding of dynamic properties in catalysis and related fields.Nanostructures with specific crystallographic planes display distinctive physico-chemical properties because of their unique atomic arrangements, resulting in widespread applications in catalysis, energy conversion or sensing. Understanding strain dynamics and their relationship with crystallographic facets have been largely unexplored. Here, we reveal in situ, in three-dimensions and at the nanoscale, the volume, surface and interface strain evolution of single supported platinum nanocrystals during reaction using coherent x-ray diffractive imaging. Interestingly, identical { Understanding strain dynamics and their relationship with crystallographic facets have been largely unexplored. Here the authors demonstrate how the 3D lattice displacement and strain evolution depend on the crystallographic facets of Pt nanoparticles during CO oxidation reaction, providing new insights in the relationship between facet-related surface strain and chemistry. Catalytic reactivity and selectivity are strongly influenced by the NP shape and are therefore facet dependent5. Shaping NPs with well defined exposed facets is an efficient strategy to enhance not only the efficiency of catalysts, but also many other physical properties such as light absorption6 or electrical conductivity7. A better understanding of these facet dependent properties is therefore required to design nanomaterials with enhanced properties. Transmission electron microscopy provides two-dimensional (2D) projected images of NPs with a resolution and sensitivity down to the atomic level10. When used in situ, the technique can even reveal time-resolved observations of shape or size changes of metal NPs during catalytic processes12. However, these observations are always challenging to make under operando conditions, in particular at elevated temperatures13 and realistic (1 atm and higher) pressures. In addition to shape-dependent catalytic activity, lattice strain14 has a considerable influence on the reactivity of metal surfaces. By modifying the distances between surface atoms, tensile or compressive lattice strain induces an upward or downward shift of the d-band centre15, resulting, respectively, in a strengthening or weakening of the bonding to adsorbed reacting species. In platinum (Pt) for instance, a 1% tensile (compressive) strain can upshift (downshift) the 5d-band by as much as ~ 0.1 eV16, dramatically affecting the surface reactivity17 and in turn the catalytic activity. Moreover, recent results indicate that metal NPs are super-strong and can sustain elastic strains in the range of several percent18. This opens a wide window for modifying the catalytic activity of the particles. Correlating surface strain with catalytic performance is therefore of fundamental importance for the development of highly efficient catalytic nanomaterials. Progress in nanomaterial design enables many opportunities for engineering the surface strain in a number of nanocatalysts by tuning structural properties such as composition, architecture (core-shell structure20) or morphology21. Applying a controlled external force on the nanocatalyst to achieve the desired strain state is also a powerful way to get a better insight in the fundamental relationship between surface strain, adsorption properties and reactivity. Several approaches have been proposed to precisely tune surface strain, they include, for instance, the physical bending or stretching of the supporting substrate22 or extracting and intercalating ions from a battery electrode support14. The latter approach has confirmed the clear relationship between enhancement or suppression of Pt oxygen reduction reaction activity under compression or tension, respectively.The chemical properties and performance of supported metallic catalysts depend on their structural properties. The demonstration of the structure-activity relationship on single crystal surfaces23. This is particularly true for NPs, where atoms located within facets or at the edges or corners have very different coordination numbers, leading to a large and complex distribution of surface strains24. Moreover, the localised nature of adsorption leads to complex site- and adsorbate-dependent responses, emphasising the need to directly study the reaction under strain, rather than extrapolating the known behaviour of individual adsorbates25. Nonetheless, studies aiming at investigating the 3D strain evolution in NPs during reaction remain relatively sparse. In particular, the strain evolution at the NP substrate interface and as a function of the nanocrystal facets is mostly unknown. In situ Bragg coherent diffraction imaging (BCDI) recently demonstrated the possibility to acquire 3D strain maps of NPs and nanostructures26. In particular, the technique has been successfully used to reveal in situ bulk and surface strain dynamics as well as localisation of active sites during reaction29.If the statement “tension strengthens binding” has gradually become a widely accepted trend, it is not a universal rulehkl} facets of NPs during reaction. The nanoscale facet dependent strain information presented here has been inaccessible to date.In this work, we demonstrate how the 3D lattice displacement and strain evolution depend on the crystallographic {α-Al2O3 (see Methods section). The sample was placed in a water-cooled gas flow reactor, in which the temperature and gas composition can be tuned  pure Ar flow, (2) Ar and O2 with O2 concentration ranging from 2% to 12.5%, (3) CO oxidation, either in reducing CO oxidation  and (4) Ar and 25% CO flow. An overview of the gas cycles carried out for each NP is available in Supplementary Tables ∘C/min in pure Ar flow was used to reach the reaction temperature of 450 ∘C. However, since several scans were measured during the heating ramp, it took 415 min to reach 450 ∘C from RT -oriented Pt nanocrystals supported on ned Fig. . The pre111 Pt Bragg reflection using an area pixel detector at an energy of 8 keV at the P10 beamline of the PETRA III synchrotron (see Methods section). Supplementary Fig. 111 Bragg peak during stoichiometric CO oxidation . The spatial resolution of the reconstructed images has been evaluated to 10.5 nm using the Phase Retrieval Transfer Function (PRTF)32  of the 111 Fig. : Es = \dϵ111 strain field measured in NP650 in Ar atmosphere and the ϵ111 strain field obtained by energy minimisation of a 200 nm NP with a similar shape. The Foiles Pt potential34, which yields the best agreement with the experimental data, was used to model the interaction between Pt atoms. In order to enable a quantitative comparison with the measured experimental surface strain field, the same voxel size (6.7 nm) was used for the experimental and simulated NPs. The latter corresponds to an average of the voxel size along the three directions. This implies that the surface strain is actually averaged over the 30 topmost surface layers. The surface strain also depends, to some extent, on the selection of the isosurface on the experimental data, as discussed in Supplementary Fig. 35. Free surface boundary conditions were used for all facets, except at the interface  (1), where angular brackets refer to average values, d is the particle diameter, and E is the isotropic Young modulus of Pt. Estimating  < σ> by the surface energy of Pt yields  < ϵ > ~ 3 × 10−4 and 1 × 10−4 for the large simulated and experimentally studied particles (NP650), respectively, in a good agreement with the strain amplitudes in Fig. 2 adsorption and CO oxidation observed in the present work  for both defect-free NPs. The cooling down to room-temperature (RT) after thermal dewetting at 1100 29. Interestingly, both strain and displacement fields are extremely stable and reproducible for a given gas condition for NP650 , making the former closer to the thermodynamical equilibrium shape of Pt41.The reaction induced strain evolution is not only observed at the interface and free surfaces but also in the volume of the NPs. As illustrated by the 2D slices of the reconstructed displacement and strain of NP300 shown in Supplementary Figs. 42. Using the facet analyser plugin43 of ParaView (see Methods), one can visualise the average surface displacement and strain per facet in 3D  are also indicated. During the first two cycles with a 2.5% O2 flow, the strain evolution is strongly dependent on the hkl Miller indices of the facets. Interestingly, all facet families except the {12 exposure, either a relaxation of the compressive strain for the {1χ = 10), where most of the facets come back to their initial strain state. Figure α-PtO2 are not stable under the reaction conditions of this work37. Rather, the facet dependent reactivity can be rationalised in terms of oxygen chemisorption during O2 exposure and oxygen reduction/desorption during reducing CO oxidation condition. Upon oxygen exposure, oxygen adsorption will occur preferentially on edge and corner atoms followed by the facet sites with increasing coordination number, n: {113} and {110} (n = 7), {100} (n = 8) and {111} (n = 9). The initial surface tensile strain in the {112 exposure at 450 ∘C. In contrast, the high coordination number of the {111} surfaces and their initial compressive strain make the binding of adsorbates on these facet sites energetically unfavourable. Binding is mostly restricted to edge and corner atoms for the {144. During reducing CO oxidation (χ = 10), the almost perfect reversibility of the strain state indicates a complete oxygen reduction, leaving only metallic Pt on the surfaces.The strain variation induced by the binding of adsorbates on the free surfaces depends on the facet type29. Even more interesting, the facet position with respect to the interface also affects its reactivity. This is illustrated in Supplementary Fig. 2 formation. Although mass spectrometry data was not collected for this sample, Supplementary Fig. 2 during CO oxidation at 450 ∘C, further suggesting that the structural changes observed during reaction are indeed linked to CO2 formation. These findings strengthen the importance of taking into account metal-support interaction to improve performance in catalysis in agreement with ref. 45. It is highly probable that CO2 formation is increased at these preferred active sites. Strain may lead to the occurrence of fluctuating surface steps acting as active sites at high temperature (450 ∘C). Reaction induced structural changes are associated to a higher reactivity and could improve the activity of the nanocatalyst.Figures nt Figs.  and 7 cont Figs. . This rent Figs.  and S8h.28. A closer look at Supplementary Table 12 suggested that diffusion of oxygen into the subsurface and bulk can lead to the formation of vacancies, which create disorder, leading to an increase of both bulk and surface strain. This diffusion of oxygen adsorbates in the lattice could be at the origin of the increase of the FWHM of the Bragg peak  with an electron beam evaporator. The Pt nanocrystals have their c-axis oriented along the [111] direction normal to the (0001) sapphire substrate. A standard photolithography method was employed to prepare a patterned layer of photoresist on sapphire prior to the electron beam evaporation of Pt. The lithographic processing route ensured that a number of dewetted Pt particles are well-separated from their neighbours and that only one crystallite is irradiated by the incoming x-ray beam.Pt nanocrystals were prepared by the solid-state dewetting of a 30-nm thin Pt film for 24 h at 1100 2 or CO). The average strain is related to the variation of the d-spacing of the (111) Pt atomic planes: 〈ϵ111〉 =  and q111 scattering vector: π/∥ q111∥ = 2π/qx, qy and qz are the coordinates of the q111 scattering vector. The conversion of pixels from the detector images into q111 coordinates was done using the xrayutilities package48. The q111 coordinates were determined by taking the centre of mass of the 3D datasets, while the FWHM of the 111 Pt diffraction patterns along the Qy direction was obtained by performing a Gaussian fit of the 3D data sets.The 3D datasets of the reciprocal space maps (RSMs) of the NP give enough information to retrieve the average strain of the measured (111) atomic planes as a function of the gas composition , outboard and vertically upward, respectively. The beam size was focused down to 0.9 μm  × 1.1 μm  using Be compound refractive lenses. The sample was mounted in a dedicated water-cooled gas-flow chamber with the substrate surface oriented in the horizontal plane on a high-precision (1 μm) XYZ-stage that was mounted on a 6-circle Huber diffractometer. The diffracted beam was recorded with a 2D EIGER X4M photon-counting detector (2070 × 2167 pixels with a pixel size of 75 × 75 μm) positioned on the detector arm at a distance of 1.839 m. We measured the 111 Pt Bragg reflection in three-dimensions by rotating the particle around the Bragg angle (20∘) through 3∘, with 0.012∘ steps for NP300 and through 2∘, with 0.005∘ steps for NP650. The counting time per point was 0.5 s. The detector was positioned at an out-of-plane angle of 39.26∘.The BCDI experiment was performed at a beam energy of 8 keV (wavelength of 1.55 Å) at the P10 beamline of the PETRA III synchrotron at DESY; 49. Phase retrieval was carried out on the raw diffracted intensity data. The initial support, which is the constraint in direct space, was estimated from the auto-correlation of the diffraction intensity. A series of 1400 Relaxed Averaged Alternating Reflections (RAAR50) plus 300 Error-Reduction (ER52) steps, including shrink wrap algorithm every 20 iterations53, were used. A support was built from the best reconstructed object. Afterwards, the support was fixed during the first 20 iterations and then let free, while reapplying a series of 600 Hybrid Input Output (HIO54) followed by 900 Relaxed Averaged Alternating Reflections plus 300 Error-Reduction steps, the shrink wrap algorithm was applied every 20 iterations. The phasing process included a partial coherence algorithm to account for the partially incoherent incoming wave front55. To ensure the best reconstruction possible, we kept only the best 50 reconstructions (with lowest free Log-Likelihood56) from 300 with random phase start and performed the decomposition into modes56. The reconstruction was then corrected for refraction using the bcdi package57. After removing the phase ramp and phase offset , the data was finally interpolated onto an orthogonal grid for ease of visualisation.The reconstructed Bragg electron density and phase were obtained using the PyNX packageγ111/γ100, γ110/γ100 and γ113/γ100. Note that different ratios were used for the two NPs, both of them differing from the theoretical surface energies for Pt39, implying that the nanocrystals did not reach their thermodynamical equilibrium shape. Several crystal sizes were considered ranging from 80 × 80 × 80 to 500 × 500 × 500 unit cells. This corresponds to a size of 32 nm/937000 atoms for the smallest configurations and 200 nm/230 x 106 atoms for the largest configurations. To obtain accurate and realistic relaxed configurations that reproduce as faithfully as possible the displacement fields measured in the experimental particle, MS simulations were carried out with the open-source Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS)58. The interaction between atoms were modelled with different embedded-atom model (EAM) Pt potentials which predict slightly different elastic properties and surface energies, parameters that are essential to model accurately the experimental displacement and strain fields. We used the EAM potentials developed by refs. 60. The NPs were relaxed at 0 Kelvin using a conjugate gradient algorithm to obtain the equilibrium displacement field.In order to model accurately the experimental NPs, several material simulation tools were used. The simulated NPs were generated using the atomistic simulation code MERLIN ), by creating a cube of atoms and cutting it along the <1 1 1>, <1 0 0>, <1 1 0> and <1 1 3> crystallographic directions, the position of the cut planes being defined by the ratio of the surface energies q is the scattering vector, fj(q) and rj are respectively the atomic scattering factor and position of atom j. The computation was performed with a GPU using the PyNX61 scattering package, which considerably sped up the calculation of the 3D diffraction patterns. The reciprocal volume over which the calculation was performed was selected based on the target voxel size in the real space data. For the largest configurations , the voxel size was equal to the experimental one (6.7 nm). The surface strain values for both NPs shown in Supplementary Table In order to allow a quantitative comparison with the experimental data, the three-dimensional diffraction patterns were calculated by summing the amplitudes scattered by each atom with its phase factor, following a kinematic approximation:u111 and ϵ111 were derived from the complex sample density r). The latter was obtained by performing a simple inverse Fourier transform of the scattered amplitude g111.u111 and the corresponding strain field ϵ111 = Finally, the real space displacement and strain fields, 43. The algorithm is based on the analysis of the probability distributions of the orientations of triangle normals of mesh representations of the particles. From the output list  of the facet recognition algorithm, we have developed a Python script to determine the hkl Miller indices of each facet as well as their average lattice displacement, strain and associated standard deviation.We have used the Facet Analyser plugin of the ParaView software, which is an automated 3D facet recognition algorithm that allows to detect the voxels at the surface of the particles and to index their facets by tuning parameters like sample size, angle uncertainty, splat radius or minimum relative facet size−6 eV between two electronic cycles. Atomic structures were relaxed until the Hellmann–Feynman forces were as low as 0.02 eV/Å. Calculations were performed using a 450 eV cutoff energy and Γ-centred 14 × 14 × 14 and 14 × 14 × 1 Monkhorst-Pack grids for bulk and (100)/(111) surface calculations, respectively. Those parameters were chosen to achieve a precision for the total energy lower than 0.1 meV/atom. In this work, the approach used to compute the adsorption of oxygen onto Pt surfaces is the typical symmetric slab mode, wherein a supercell of the crystal oriented to expose its (hkl) surface is generated, and atoms are removed from a portion of the supercell to create a vacuum . The atomic strain tensor for each atom in the slab was extracted using the atomic strain modifier in OVITO62, considering a cut-off radius of 3.09 Å. This value corresponds to the first minimum of the pair distribution function for Pt, i.e., halfway between the first and the second shell of neighbours. To compare with the experimental values, the atomic strain values were averaged over the size of the slab along the z-direction (20 Å). Since the average surface strain mostly scales with the voxel size, this value was then multiplied by the ratio between the size of the slab and the experimental voxel size (67 Å). Finally, to allow a quantitative comparison with the experimental values 〈ϵ111〉 of the {1ϵ111〉 values have to be multiplied by cos2α where α is the angle between q111 and the facet normal , within the projector-augmented wave method (PAW), for the description of the interaction between the valence electrons and the ionic core. The GGA-PBE functional was used, with DFT-D3 corrections. We consider atomic valences to be 2s2 2p4 (O) and 5d9 6s1 (Pt). Total energies were minimised until the energy differences were less than 1 × 10Supplementary InformationPeer Review File


Angle-resolved photoelectron spectroscopy (ARPES) performed on the vicinal SmB6(001)-p(2 × 2) surface showed that TSSs are anisotropic and that the Fermi contour lacks the fourfold rotation symmetry maintained in the bulk. This result emphasizes the important role of the surface atomic structure even in TSSs. Moreover, it suggests that the engineering of surface atomic structure could provide a new pathway to tailor various properties among TSSs, such as anisotropic surface conductivity, nesting of surface Fermi contours, or the number and position of van Hove singularities in 2D reciprocal space.The topology and spin-orbital polarization of two-dimensional (2D) surface electronic states have been extensively studied in this decade. One major interest in them is their close relationship with the parities of the bulk (3D) electronic states. In this context, the surface is often regarded as a simple truncation of the bulk crystal. Here we show breakdown of the bulk-related in-plane rotation symmetry in the topological surface states (TSSs) of the Kondo insulator SmB Previous work exploring the robustness of topological surface states to perturbations has mostly focused on surfaces with the same atomic structure as the bulk. Here the authors demonstrate the effect of surface reconstruction on the topological surfaces on the (100) surface of SmB6. Such topological surface states (TSSs) are attractive not only for basic science but also for spintronic applications due to their momentum-dependent spin–orbital polarization3. Since TSSs originate from the topological order of bulk states, some qualitative characteristics of TSSs, such as the metallic band dispersion across the bulk bandgap, are robust against any non-magnetic external perturbations as long as the bulk states remain unchanged. This is a strong point on the one hand because of the expected contamination-tolerant working of devices, for example. On the other hand, the role of the surface atomic structure in TSSs is regarded as rather unimportant because of the robustness. Some theoretical and experimental studies reported modification of TSSs by surface treatments, such as surface oxidization4, chemical or photochemical ageing6, and a small interlayer rotation forming a moiré modulation on the surface7. However, such previous works were focused on how to perturb the pristine TSS. Actually, the unit cells of the surface lattice in these works were always the same length of basis vectors as the ideally truncated bulk crystal, with the symmetry operations obtained by simple projection of bulk 3D space group. Some studies focused on topological electronic states localized at 1D edges or hinges, but they still suppose the similar simple projection from 3D to 1D systems9. Although some drastic electronic phenomena, such as 1D charge-density-wave formation on 2D semiconductor surfaces10, obtained by forming a surface atomic structure independent of the substrate, are already known, the role of the surface superstructures in TSSs has not been studied in detail yet, to the best of our knowledge.The close correspondence between symmetry operations in 3D bulk bands and the topological character of the surface states lying on the edge of a crystal has been one of the central topics of solid state physics in this decade6) is a long-known Kondo insulator, in which a bulk bandgap opens at low temperature because of the Kondo effect11. It is the first material proposed as a candidate for topological Kondo insulators (TKIs), and it hosts a metallic TSS coexisting with strong electron correlation13 and was recently confirmed as a TKI by angle-resolved photoelectron spectroscopy (ARPES) experiments after a long debate18, as summarized in ref. 6 is also known as a valuable example of a well-defined surface superlattice among TIs. Although cleavage provides multiple surface terminations20, in situ surface preparation, typically by the cycling of Ar ion sputtering and annealing, results in an uniform surface superstructure such as (1 × 2) and (2 × 2)22, making the SmB6(001) surface a good template to study the role of the surface atomic structure in TSSs. The remaining barrier for such research is that the obtained SmB6(001) surfaces have two or more equivalent domains coexisting with nearly the same area. For example, double-domain (1 × 2) and (2 × 1) surfaces could provide apparent fourfold rotation symmetry of the TSS by overlapping of two twofold domains rotated 90° to each other, as observed on the Si(001) surface23.Samarium hexaboride (SmB6(001)-p(2 × 2) surface prepared in situ. One surface domain is dominantly grown by using a vicinal (001) substrate, confirmed by low-energy electron diffraction (LEED). The dispersion and orbital–angular-momentum (OAM) polarization of the SmB6(001) TSS is observed by ARPES without ambiguity from the multidomain overlap for the first time. The ARPES data show that the TSS is anisotropic and that the Fermi contour (FC) lacks the fourfold rotation symmetry that is maintained in bulk. This result emphasizes the important role of the surface atomic structure even in the TSS.In this work, we show the TSS on the uniform and semi-single-domain SmB6 has a fourfold rotation symmetry. After cycles of Ar ion sputtering and annealing (see “Methods” for details), the vicinal SmB6(001) surface exhibited the LEED patterns shown in Fig. 21, the (1/2 1/2) fractional spot indicated the p(2 × 2) surface structure at the primary electron energy (Ep) of 65 eV. Moreover, other fractional spots at Ep = 45 eV, (1 − 1/2) and (1/2 − 1), show different intensities from each other, although they should be identical if the surface has fourfold rotation and time-inversion symmetries. Note that different diffraction spots become bright at Ep = 45 and 65 eV simply because to sweep LEED Ep corresponds to sweep the size of the Ewald sphere in reciprocal space. The asymmetric intensities between (1 – 1/2) and (1/2 – 1) are quantitatively shown by the LEED line profiles in Fig. p(2 × 2), and thus both (1 − 1/2) and (1/2 − 1) spots could appear even from the perfect single-domain surface.As depicted in Fig. al SmB600 surface al SmB600 surface s core level and Sm2+ 4f valence bands obtained from angle-integrated photoelectron spectra of SmB6(001)-p(2 × 2). Different from the cleaved SmB6(001) cases17, we found no clear “surface” term which appears at different energies from the main peaks. This would occurs because the heating process during the surface preparation removes the metastable Sm and B atoms which are the origin of the “surface” terms. The B 1s peak has a tailed shape with respect to higher kinetic energies, suggesting that some boron atoms are in a different surrounding environment from the bulk case, but the difference is not as drastic as in the "boron-terminated” case of the cleaved surfaces20. This suggests that small displacements of the surface boron atoms are the origin of the p(2 × 2) superlattice. While some cleaved surfaces of SmB6(001) were reported to be inhomogeneous with various termination atoms24, we found no such signature from the current surface. Further discussion on this point is shown in Supplementary Note Figure of SmB600-p(2 × 2)p(2 × 2) surface superstructure, as illustrated in Fig. p(2 × 2) superlattice is formed only by displacements of B6 and Sm atoms at the topmost two atomic layers. Such picture agrees with the less drastic change of surrounding conditions of the surface atoms, which was suggested by the core-level spectra explained above. In addition, the number of neighboring atoms of the displaced Sm is the same as those in bulk, in contrast to the reduction for the topmost B6. It rationalizes the core-level spectra showing the "surface” term only for boron peaks. Note that this model is just a possible example to satisfy what were observed by LEED and core-level spectra. Further experiments are required to determine the surface atomic structure accurately, such as dynamical LEED analysis25 or Weissenberg reflection high-energy electron diffraction26, while the p(2 × 2) surface unit cell without fourfold rotation symmetry is enough for the following discussion in this article.Based on these results, we propose a possible model of EF) measured with circularly polarized photons at 35 eV. The spectra obtained by using both right- and left-handed polarizations are summed to avoid the anisotropic intensity distribution due to the circular dichroism (CD). Figure 21. The stark contrast to the earlier studies is that the FC around the other ky//[010] orientations are identical to each other, even when the photoelectron incidence orientation is taken into account. Moreover, S1’ becomes as evident as S1 near the edge of the crystal surface where the vicinal miscut is expected to be different from that at the center of the polished surface, indicating that the difference between S1 and S1’ is from the surface domains (ARPES data are shown in SM). Together with the anisotropic LEED patterns -p(2 × 2) surface lacks the fourfold rotation symmetry with the S1 FC only around one Figure entclass1pt{minimaentclass1pt{minimaentclass1pt{minima16. This is due to the uniform single-domain surface preparation over a wide area of the sample. The shape of S2 is similar to that observed on the cleaved (001) surface. While that state was claimed as the umklapp of S1 by (1 × 2) surface periodicity at first17, this assignment has not yet reached a consensus, as the following discussion made a counterargument on this assignment19. On the other hand, as shown in Fig. The other FCs enclosing entclass1pt{minimaf and 5d orbitals. Such condition can be naturally satisfied by assuming p(2 × 2) surface superstructure; for example, minor displacements of Sm below the topmost surfaces as depicted in Fig. All the three FCs observed here lacks the fourfold symmetry, as shown in Fig. ssuming p × 2 surfEF. The almost localized Sm 4f band lies slightly below EF (~20 meV) and the Sm 5d bands disperses in 150–50 meV, reflecting the itinerant character. Around the crossing point between them, the bands bend, indicating the c-f hybridization induced by the Kondo effect. Above the Sm 4f band, the metallic bands continuously disperses between EF and Sm 4f in Cuts 1–3. Their Fermi wavevectors (kF) are consistent with the FCs observed in Fig. EF along this orientation. While the surface bands corresponding to S2 and S3 are not very clear from the 2D intensity plot in Fig. f band and the momentum distribution curve (MDC) at EF shows distinct peaks corresponding to them. Moreover, Fig. EF without such difficulty. This is thanks to the photon energy (18 eV) for Fig. f becomes small. The kF values for S2 and S3 obtained in Fig. EF with different photon energies  point; thus, the polarization of photoelectrons is not always the direct information of the initial state30. Therefore, further datasets, especially spin-resolved ARPES with multiple incident-photon energies and polarizations, are required to conclude the winding number of the TSS.Figure 6(001)-p(2 × 2) is highly anisotropic. From this anisotropy, the other electronic properties, such as the electron conductivity and net spin polarization via the surface currents, are also suggested to be anisotropic on SmB6(001)-p(2 × 2), as far as they are derived from the TSS. Note that none of these properties are topologically protected. Therefore, examining the rotation asymmetry from the vicinal SmB6(001) sample with in situ surface preparation would be an interesting new pathway to distinguish the origin and dimensionality (2D or 3D) of unconventional electronic phenomena observed in SmB6, such as the thermodynamic properties31 and quantum oscillations37. If one could apply the surface preparation method similar to ours to vicinal SmB6(001) sample and then insert it to a magnet equipment for de Haas van Alphen oscillation measurements without breaking the vacuum environment, such experimental setup would provide conclusive evidence about the role of surface on the quantum oscillation.We have revealed that the TSS of SmB6(001)-p(2 × 2), surface superstructure causes no change the energetic order of bulk bands nor magnetic order. Therefore, there shouldn’t be any change in topological order before and after the formation of surface superstructure. However, it would be helpful to discuss how to determine the topological order with the surface superstructure, since it is often determined by counting the number of FCs to be odd or even, around surface time-reversal invariant momenta (TRIM); 6(001)38. For this counting, we propose to double check the number of FCs both by using (1 × 1) bulk-truncated SBZ as well as that with folding according to the surface superstructure. Circled numbers in Fig. 6(001)-p(2 × 2), showing 3 FCs, odd number consistent with the non-trivial topological order19. Note that S1 at the bottom side of Fig. Since the folding of FCs by surface superstructure changes the apparent shape of FCs drastically, one could have a question about its role on topological order. In most cases including SmB40. Moreover, this work exhibited that the surface superstructure distorts surface FCs at the same time. It suggests that engineering of surface superstructure could tailor the number and position of VHS in the reciprocal space as well as the other parameters of FCs, such as nesting vector dominating surface density-wave formation41. Actually, the observed FC on SmB6(001)-p(2 × 2) does exhibit triple degenerate points of TSSs as guided by arrows in Fig. 43. From these perspectives, detailed electron behavior in smaller energy and temperature scale, in orders of few meV and Kelvins, respectively, of SmB6(001)-p(2 × 2) would be an encouraging test peace to examine the role of the surface superstructure on TKI.The folding of TSS by surface superstructure could play further role on the unconventional electronic phenomena of TSS. It duplicates FCs formed by TSS, making the additional crossing points with van-Hove singularity  plane was mechanically polished in air with a small angle offset toward [010] until a mirror-like shiny surface was obtained with only a few scratches when observed under an optical microscope (multiple ×10 magnification). The miscut angle is estimated to be ~ 1° from the side-view images obtained by the optical microscope. The polished sample was moved to ultra-high-vacuum (UHV) chambers and cleaned in situ by repeated cycles of Ar ion sputtering (1 keV) and annealing up to 1400 ± 50 K. The cleaned surface was transferred to the measurement chambers for LEED or ARPES without breaking the UHV.Single crystalline SmBThe LEED measurements were performed by using a conventional electron optics  at beamline 7U of UVSOR-III. The sample was mounted on a homemade 6-axis goniometer maintained at 100 K. The electron incident angle was adjusted to be parallel to the surface normal ([001]) by using the rotation angles of the goniometers.EF and the overall energy resolution of the ARPES setup ( ~ 15 meV) were calibrated using the Fermi edge of the photoelectron spectra from Ta foils attached to the sample.The ARPES measurements were performed with synchrotron radiation at the CASSIOPÉE beamline of synchrotron SOLEIL. The photon energies used in these measurements ranged from 18 to 240 eV. The polarization of incident photons is depicted in Fig. Supplementary InformationPeer Review File


Electrical control of those correlated electronic states and AHE allows us to resolve their own nature and origin and to discover new quantum phenomena. Here, we show that electrically controlled proton intercalation has significant impacts on striking quantum phenomena in CsV3Sb5 nanodevices mainly through inducing disorders in thinner nanoflakes and carrier density modulation in thicker ones. Specifically, in disordered thin nanoflakes (below 25 nm), we achieve a quantum phase transition from a superconductor to a “failed insulator” with a large saturated sheet resistance for T → 0 K. Meanwhile, the carrier density modulation in thicker nanoflakes shifts the Fermi level across the charge density wave (CDW) gap and gives rise to an extrinsic-intrinsic transition of AHE. With the first-principles calculations, the extrinsic skew scattering of holes in the nearly flat bands with finite Berry curvature by multiple impurities would account for the giant AHE. Our work uncovers a distinct disorder-driven bosonic superconductor-insulator transition (SIT), outlines a global picture of the giant AHE and reveals its correlation with the unconventional CDW in the AV3Sb5 family.The electronic correlations , chiral charge order and nematic order) and giant anomalous Hall effect (AHE) in topological kagome metals AV 3Sb5 nanoflakes, and show its effect on superconductivity, charge density wave and anomalous Hall effect.Kagome metals continue to attract interest due to the coexistence of electronic correlations and band topology. Here the authors use proton gating to modulate disorder and carrier density in CsV This is in no small part due to the many quantum phenomena that they support including unconventional SC11, novel nematic order12, chiral charge density order22, giant anomalous Hall effect25 as well as the interplay between two-gap SC and CDW in CsV3Sb526. The unique coexistence of electronic correlations and band topology in AV3Sb5 allows for investigating intriguing transitions of these correlated states, such as the superconductor-insulator transition (SIT), a protocol quantum phase transition (QPT) that is usually tuned by disorders, magnetic fields and electric gating28. Moreover, the origin of giant AHE in AV3Sb5 and its correlation with chiral CDW remain elusive30, in spite of several recently proposed mechanisms including the extrinsic skew scattering of Dirac quasiparticles with frustrated magnetic sublattice23, the orbital currents of novel chiral charge order13 or the chiral flux phase in the CDW phase31. Thus the ability to tune the carrier density and the corresponding Fermi surfaces would play a vital role in understanding and manipulating these novel quantum states and further realizing exotic QPTs.The layered kagome metals AV33 exhibits crucial impacts on the superconducting state, CDW state and the associated AHE in CsV3Sb5 nanoflakes via disorders and carrier density modulation. In thinner nanoflakes (below 25 nm) with large gate voltages (e.g. with an amplitude above 15 V), the enhanced disorders from intercalated protons suppressed both CDW and superconducting phase coherence and gave rise to a SIT associated with the localized Cooper pairs, featuring a saturated sheet resistance reaching up to 106 Ω for T → 0, dubbed a “failed insulator”. While in thicker CsV3Sb5 nanoflakes with much lower gate voltages (within 7 V), the superconducting transition instead retained with nearly unchanged sheet resistance in normal state at 5 K, indicating very limited impact of disorder. However, the Hall measurements demonstrate a large modulation of carrier density (with the modulation up to 1022 cm−3) and the relevant Fermi surface topology changes from a hole pocket to an electron pocket. Consequently, we find that the giant anomalous Hall conductivity (AHC) with a maximal amplitude exceeding 104 Ω−1 cm−1 mainly pinned down to a narrow hole-carrier-density window around In this work, we find that electrically controlled proton intercalation3Sb5 has a hexagonal crystal structure with space group P6/mmm (No. 191). As shown in the upper panel of Fig. 3Sb layers are sandwiched by antimonene layers and Cs layers. X-ray diffraction (XRD) . Next we demonstrate how the proton intercalation significantly affects the SC state, CDW state and the giant AHE in CsV3Sb5 nanoflakes with distinct thicknesses.The layered material CsV/mmm No. 91. As sh3Sb5 flakes below 25 nm. Figure s − T curve  with l being the thickness of a single layer, we get s reaching up to 106 Ω on insulating side, however, a saturated resistance trend appeared for T → 0 K, as shown in Fig. 37. This type of superconductor to “failed insulator” transition has also been observed in another sample #8 in Supplementary Fig. 38.We first investigate the impacts of proton intercalation on the correlated electron states including SC and CDW in thinner CsV42. To get the critical exponents of SIT, we plot more than twenty sets of Rc suggests the bosonic nature of SIT, in which the coherent Copper pairs in the SC phase are localized by disorders with loss of macroscopic phase coherence in the insulating phase28. The finite size scaling dependence of Rs on T and a tuning parameter has the form s is the charge density, nc is the critical carrier density with the value of 0 is the scaling parameter which approaches to zero at ns = nc. ν is the correlation-length exponent and z is the temporal critical exponent42. By extracting the exponent product νz and plotting lnT0 versus 43, which was also attributed to localization of persisting Cooper pairs. Note that the critical exponent here is distinct from those of 2D conventional models for SIT28 such as classical percolation model (vz = 4/3), quantum percolation model (vz = 7/3), which probably results from the complexity of SC gap and multiple impurities44. Thus, CsV3Sb5 provides us with a unique example system to explore rich QPTs involving intrinsic superconductors with topological energy bands and frustrated kagome lattice.SIT can be usually characterized by two distinct scenarios (bosonic and fermionic) according to the nature of insulating phase, while the finite-size scaling analysis could yield its critical exponents and further reveal the universality class of QPTg. Interestingly, CDW transition temperature 3Sb5 under high pressures, possibly due to band reconstructions or Fermi level shift8. It is clear that the protonic gate significantly modifies the CDW and SC phases, facilitating the further investigation of the intertwinement among these novel electronic correlations in AV3Sb5.Figure 3Sb5 nanoflakes with much lower gate voltages within 7 V and find that the proton intercalation mainly changes the carrier density in those thicker ones, leading to only a slight modulation of SC transition temperature  × 1022 cm−3 with the AHC around 5 × 103 Ω−1 cm−1. Shifting away from these two regions, however, the AHC either keeps a finite value or approaches zero for devices #1 and #2  and intrinsic regimes (at Vg = −4.6 V) reveals a strong dependence of AHE on the Fermi energy of CsV3Sb5.The scaling law between AHC and σ45, the realistic intrinsic AHC from field-induced magnetization should be much smaller than the observed intrinsic AHC. It has been known that the extrinsic skew scattering of AHE essentially originates from the asymmetric scattering of carriers by nonmagnetic/magnetic impurities. Usually, there are three distinct scenarios to produce the extrinsic skew scattering and the resultant AHE49. By careful examination of each scenario, we could exclude the Kondo scattering and resonant skew scattering  doping can significantly shift the van Hove singularity (VHS1) upward (downward) with respect to the Fermi level within the rigid-band approximation, as shown in Fig. 3Sb5 single crystal, the Fermi level lies slightly above VHS1 near the M point , exhibiting a large AHE. In exfoliated CsV3Sb5 nanoflakes, the Fermi level approaches the lower sub-band due to the increasing Cs vacancies and the AHC at Vg = 0 V reduces to about one third of the maximal value, i.e., 4500 −1 cm−1. Applying a negative voltage will accordingly lower the Fermi level (details in Supplementary section −1 cm−1) in the electron pockets. At Vg > 0 V, however, the Fermi level will be shifted upward and back to the upper sub-band again (dashed red arrow), the giant AHE reappears at 4.5 V in device #4. This giant AHE primarily comes from the skew scatterings of holes in the nearly flat bands at VHS1 with nonzero Berry curvature by the V vacancies and/or PM impurities. The large discrepancy of AHE in the electron and hole pockets is consistent with asymmetric distribution of DOS in the CDW bands near the M points11. Note that the intrinsic AHC at Vg = −4.6 V near the electron-hole crossover point is mainly ascribed to the large suppression of the DOS at the middle of the CDW gap. After evaluating the possible AHE from the electron near the Γ point and the Dirac bands, we find this intrinsic AHE mainly originates from the recent chiral charge order forming from the electronic states near the saddle point61.We further investigate the impact of charge doping on the band structure and AHE. Since the charge doping in CsV3Sb5, inducing disorders in thinner nanoflakes and carrier density modulation in thicker ones. In thin nanoflakes below 25 nm with 3Sb5 can be attributed to the intense extrinsic skew scattering of holes in the nearly flat bands with finite intrinsic AHE in the CDW phase at the saddle points by multiple impurities. This significant and electrically controlled SIT and AHE in CsV3Sb5 should inspire more investigations of the relevant intriguing physics and promising energy-saving nanoelectronic devices.In summary, we revealed two major impacts of proton intercalation on CsV3Sb5 were synthesized via Sb flux method. The elemental Cs, V and Sb were mixed at a molar ratio of 1:3:20, and loaded in into a MgO crucible. This process was performed in a glove box in Ar ambience. Then the crucible was sealed in a vacuumed quartz tube. The ampule was slowly heated to 1000 ˚C and kept for 20 h. After cooling at a rate of 2 °C/min, the extra flux was removed by fast centrifuging at 640 °C. , ethanol, deionized water, phosphoric acid  with a typical molar ratio 1:18:6:0.03. The mixed solution was then stirred for 2 h and annealed for another 2 h at 50 °C in a sealed bottle to form polymerized Si–O–Si chains. Finally, the substrate with bottom gate electrodes was spin-coated with the prepared protonic solution and baked at 150 °C for 25 mins. Transport measurements were performed in a commercial Physical Property Measurement System (PPMS) with magnetic field up to 9 T and a commercial magnetic property measurement system (MPMS) with magnetic field of 7 T.Supplementary InformationPeer Review File


Scientific and public interest in the potential ergogenic effects of sodium alginate added to a carbohydrate (CHO) beverage has increased in the last ~ 5 years. Despite an extensive use of this technology by elite athletes and recent research into the potential effects, there has been no meta-analysis to objectively elucidate the effects of adding sodium alginate to a CHO beverage on parameters relevant to exercise performance and to highlight gaps in the literature.Three literature databases were systematically searched for studies investigating the effects of sodium alginate added to CHO beverage during prolonged, endurance exercise in healthy athletes. For the systematic review, the PROSPERO guidelines were followed, and risk assessment was made using the Cochrane collaboration’s tool for assessing the risk of bias. Additionally, a random-effects meta-analysis model was used to determine the standardised mean difference between a CHO beverage containing sodium alginate and an isocaloric control for performance, whole-body CHO oxidation and blood glucose concentration.n = 5 studies; I2 = 0%), CHO oxidation  and blood glucose concentration . When compared with an isocaloric control, the meta-analysis demonstrated that there is no difference in performance , CHO oxidation  and blood glucose concentration  when ingesting a CHO beverage containing sodium alginate. The systematic review revealed that several of the included studies did not use sufficient exercise intensity to elicit significant gastrointestinal disturbances or demonstrate any ergogenic benefit of CHO ingestion. Risk of bias was generally low across the included studies.Ten studies were reviewed systematically, of which seven were included within the meta-analysis. For each variable, there was homogeneity between studies for performance (This systematic review and meta-analysis demonstrate that the current literature indicates no benefit of adding sodium alginate to a CHO beverage during exercise. Further research is required, however, before firm conclusions are drawn considering the range of exercise intensities, feeding rates and the apparent lack of benefit of CHO reported in the current literature investigating sodium alginate. The current empirical evidence indicates no benefit of adding sodium alginate to a carbohydrate beverage.Much of the current literature does not use sufficiently high carbohydrate intake or exercise intensity to elicit gastrointestinal distress in the control conditions, and thus, the effect of sodium alginate on gastrointestinal comfort is not well studied.−1 during exercises lasting 1–2.5 h and up to 90 g hr−1 during exercise lasting longer than 2.5 h  OR “carbohydrate”) AND (“exercise” [Mesh] OR “exercise” OR “exercise Test” [Mesh] OR “exercise tolerance” [Mesh]) AND , Web of Science: (ALL = (carbohydrate) OR ALL = (carbohydrates)) AND (ALL = (exercise) OR ALL = (exercise test) OR ALL = (exercise tolerance)) AND (ALL = (hydrogel) OR ALL = (hydrogels) OR ALL =  OR ALL = (pectin)) and SPORTDiscus: (TX carbohydrates OR TX carbohydrate) AND (TX exercise OR TX exercise test OR TX exercise tolerance) AND . The reference list of each study identified was scrutinised for any further studies to be included within the analysis. The strategic search and study selection were performed independently by two researchers (SS and BMP), with any discrepancies in included studies resolved by consultation of AB. The number of studies was identified, and their selection process is presented in Fig. The inclusion criteria for the meta-analysis focussed on the population characteristics, on the study design, on the exercise test, on the beverage characteristics, and on the main outcomes: 1) population: male or female athletes, between 18 and 50 years old, 2) study design: randomised allocation/order in a crossover design, using a control beverage, 3) exercise test: a bout of prolonged endurance exercise of at least 60 min , and 4) beverage characteristics: subjects ingesting a CHO beverage with sodium alginate and an isocaloric control beverage containing the same composition of CHO without sodium alginate. These athletes had to be healthy, with no history or presently showing symptoms of gastrointestinal disease.Those studies that failed to meet the criteria for meta-analysis, but included CHO beverage ingestion with sodium alginate and a prolonged endurance exercise with measurements of physiological variables , were included within the systematic review.After an initial screening and removal of duplicates, the titles and abstracts were assessed for suitability. The full texts of the remaining studies were examined, and a final list of studies meeting the inclusion criteria created. The following data were extracted from these selected studies: (1) title, type of publication , information on publication ; and (2) participant characteristics, study design, control group (randomisation and beverage composition), experimental group  and outcomes .−1 and blood glucose concentration as mMol L−1. Blood glucose concentration data that were presented as mg dL−1 were converted to mMol L−1 by multiplying the value by 0.0555. If data could not be extracted through the text, tables or figures, the corresponding author was contacted.Data extraction for the meta-analysis was collated, primarily through text and tables, and if the data were present in a figure only, the graph was digitised using an in-built measuring tool  with the means and SD measured manually, using the scale provided in the figure. In studies where data were reported across numerous time points, data were averaged to calculate a single mean and SD. Substrate oxidation is presented as g minIndependently, two researchers (SS and BMP) used the Cochrane Collaboration’s tool for assessing the risk of bias  and deteI2 statistic and the chi-square test [p ≤ 0.05.Review Manager  was useare test . The MD The flow chart presented in Fig. Of the 10 studies included within the systematic review, four used cycling –27, five−1; and females: age 24 ± 4 yrs, body mass 64.8 ± 7.3 kg, −1). Other participant variables were not uniformly reported across studies and therefore not reported.A total of 92 participants were included across the 10 studies included for the systematic review . No SD data were provided in two studies , 32, andOf the 10 included studies, six involved prolonged, continuous treadmill running for an average of 129 min (range 105—180 min) at an average intensity of 61% V˙O2max) , 28–32. V˙O2max) –27, 29. }V˙O2max ) and 45%}V˙O2max . Finally}V˙O2max .−1 .–29.−1  and no difference in performance between CHO beverages with or without sodium alginate and pectin  and no differences in CHO oxidation rate when ingesting a CHO beverage with or without sodium alginate and pectin  and low heterogeneity , suggesting that the current meta-analysis provided an accurate quantitative representation of the effects of sodium alginate and pectin added to CHO beverages.The present systematic review and meta-analysis has reviewed all the relevant literature investigating the use of CHO beverages with additional sodium alginate and pectin during exercise. It was found that, based on published studies, the addition of sodium alginate and pectin confers no significant benefit compared to an isocaloric beverage on performance, CHO oxidation or blood/plasma glucose. Each comparison revealed excellent homogeneity of results  endurance exercise used a relatively low intensity (~ 60% V˙O2max) , 28, 29.arathons  and mustarathons  Fig. 3).V˙O2max; , 25, 29)V˙O2max; ) to vari sprints ) intensiInitial, mechanistic studies have demonstrated that the pathway of CHO ingestion/intestinal absorption may differ from that of a “standard” CHO beverage when compared with a CHO beverage containing sodium alginate and pectin (i.e. formation of hydrogel in the stomach and faster early GE rate) , 12. How−1 of CHO with additional sodium alginate and pectin [Regarding whole-body CHO oxidation rates, Achten et al. found significant differences when comparing different types of CHO such as isomaltulose and sucrose . Specifid pectin .Despite showing no significant differences in fat oxidation between a CHO beverage or one containing sodium alginate and pectin, Barber et al. highlighted that the ingestion of sodium alginate and pectin was associated with a significant increase in the contribution of fat oxidation to total energy expenditure, resulting in a reduced endogenous CHO utilisation . Once thThe ExCHO oxidation rate resulting from the ingestion of CHO with the addition of sodium alginate and pectin has been investigated by several authors , 31, 33,The maintenance of blood glucose concentration is considered one of the major ergogenic features of CHO ingestion during prolonged exercise. The ingestion of CHO with the addition of sodium alginate and pectin has, in line with the other physiological variables measured, been shown by this meta-analysis to have no impact upon blood glucose concentrations during exercise. This finding is unsurprising considering the “downstream” variables that are directly influenced by blood glucose concentration (i.e. performance and substrate oxidation) which were found to be unaffected by the ingestion of a CHO beverage with additional sodium alginate and pectin , 28–30. recently .−1), exercise intensities (45–71% Initial reports into the use of sodium alginate and pectin to enhance CHO beverage ingestion have suggested that it may reduce the occurrence of GID through differential sensing of CHO within the intestine . Nonetheicipants , 28–30. icipants , 26, 28,icipants  or the licipants , 29. Dueicipants , the visicipants . Howevericipants  was muchThis systematic review and meta-analysis have demonstrated that the current literature shows that there are no significant differences between CHO beverages with or without sodium alginate and pectin when considering performance, substrate oxidation or blood glucose concentration during low (i.e. 45%