It shows a record low overpotential of 178 mV at 10 mA cm−2 and maintains the excellent performance throughout the 10 h chronopotentiometry test at a constant current of 10 mA cm−2 in 0.5 M H2SO4 solution. Density functional theory calculations further revealed the intrinsic mechanism for the exceptional oxygen evolution reaction performance, highlighting the influence of chromium promoter on the enhancement in both activity and stability.The development of active, acid-stable and low-cost electrocatalysts for oxygen evolution reaction is urgent and challenging. Herein we report an Iridium-free and low ruthenium-content oxide material (Cr 2 generated from water electrolysis is a promising, carbon-neutral fuel, but sluggish oxidation kinetics and poor catalyst stability in acid limits the chemistry. Here, authors prepare solid-solution catalysts based on ruthenium-chromium oxides that are stable and active for acidic water oxidation.H Essentially, the process of OER is a four electron and four proton coupled electrochemical reaction, demanding a higher energy  to overcome the kinetic barrier than the hydrogen evolution reaction (HER), which is a two electron-transfer reaction. In the past decades, substantial research effort has been devoted to the design and development of OER electrocatalysts with enhanced electrode kinetics and stability. To date, various OER catalysts, such as transition metal oxides6, perovskite8, and layered structure materials10, have been reported. However, these OER electrocatalysts still suffer from sluggish kinetics and/or low stability in acidic media. Compared with alkaline conditions, OER catalysis under acidic conditions is much more preferable because acidic electrolyte has higher ionic conductivity and fewer unfavorable reactions12. In addition, commercially available water electrolysis assemblies use cation exchange membrane, e.g., Nafion, as the ionic conductor, which requires OER to be operated in acidic environment. Unfortunately, most of the known active metal oxides cannot survive under harsh acidic operating conditions. Currently, rutile-structured ruthenium (Ru) and iridium (Ir) oxides are the two best catalysts for OER in acidic media15. It is widely accepted that RuO2 has higher activity but lower stability than IrO218. Thus, to develop OER catalysts with both high activity and stability, the use of mixed phase or solid solution of RuO2 and IrO2 has been investigated22. Very recently, three new types of Ir-based double perovskites23, multiphase IrNiOx or IrOx/SrIrO325, and pyrochlores-structured Ir-based oxides26, were reported to be active and relatively stable toward OER in acidic media. However, we note that little attention has been paid to design cheaper Ru-based electrocatalysts, particularly with low Ru-content, for OER in acidic condition27. Indeed, it is desirable to modulate the electronic structure by replacing part of Ru with suitable transition metals in order to improve the OER activity. Furthermore, the replacement by cheaper transition metal is also advantageous in terms of cost.Oxygen evolution reaction (OER) or the water oxidation plays a key role in clean energy technologies, including hydrogen production through water electrolysis, electrochemical or photoelectrochemical CO28. In light of these successful studies, we propose to design Ru-based electrocatalysts based on MOF templates, which can make use of the porosity to load Ru sources and the original metal node as promoter. Herein, we present a low-cost Ir-free rutile-structured chromium-ruthenium oxide electrocatalyst  derived from MIL-101 (Cr) which exhibits record low overpotential and high stability toward OER in acidic media. We chose MIL-101 (Cr) because of its ultra-high surface area (above 3000 m2 g−1) and large pore sizes (2.9–3.2 nm) that can facilitate the loading of Ru precursors29. Moreover, density functional theory (DFT) calculations suggested that Cr plays a critical role in improving the stability and OER activity by tuning the electronic structure of RuO2 phase. The resulting Cr0.6Ru0.4O2 electrocatalyst exhibits an overpotential of 178 mV at 10 mA cm−2, a small Tafel slope (58 mV dec−1), and stable chronopotentiometric performance under 10 mA cm−2 in 0.5 M H2SO4 solution for 10 h, which outperforms the most active OER electrocatalysts reported to date, such as BaYIrO623, IrOx/SrIrO325, and Y2Ru2O7-δ27.Metal-organic frameworks (MOFs), a unique type of porous materials with ultrahigh porosity, tunable pore sizes and morphology, and well-characterized crystalline architectures, have emerged as excellent templates or platforms for preparing electrocatalysts with high performance, such as N-doped porous carbon, metal oxide nanocomposites3-MIL-101(Cr) precursor and Cr0.6Ru0.4O2 powders is illustrated in Fig. 3 was firstly loaded into the pores of MIL-101 (Cr) by means of impregnation. After loading RuCl3, the color of MIL-101 (Cr) changed from light green to brown (the color of RuCl3)  composite was further annealed under air at temperatures between 450 and 600 °C for 4 h to fabricate Cr0.6Ru0.4O2 powders. RuCl3-MIL-101 (Cr) was characterized using a combination of power X-ray diffractions (PXRD), scanning electron microscopy (SEM), inductively coupled plasma-mass spectroscopy (ICP-MS) and N2 adsorption/desorption measurements at 77 K. As shown in Fig. 3-MIL-101 (Cr) was essentially identical to that of original MIL-101 (Cr), suggesting that the crystalline structure of MIL-101 (Cr) was preserved after loading RuCl3. The reduced intensity of the peaks below 7° after loading RuCl3 can be attributed to the pore filling of MIL-101 (Cr), which has also been observed in PEI incorporated MIL-101 (Cr)30. There was no peak for RuCl3, indicating that RuCl3 did not crystalize in the pores of MIL-101 (Cr) but was adsorbed on the pore surface. SEM characterization was conducted to analyze the morphology of MIL-101 (Cr) before and after loading RuCl3. As displayed in Fig. 3 into MIL-101 (Cr) pores. After loading RuCl3, morphology change of MIL-101 (Cr) was not observed. ICP-MS was employed to evaluate the loading amount of RuCl3 in MIL-101 (Cr). The measured atomic ratio of Cr/Ru was 6:4, corresponding to 37.8 wt% RuCl3 content in RuCl3-MIL-101 (Cr). N2 adsorption/desorption measurements of MIL-101 (Cr) and RuCl3-MIL-101 (Cr) were further conducted to evaluate their surface area and pore volume  surface area were decreased to 0.97 cm3 g−1 and 1783 m2 g−1, respectively.The route to the preparation of RuCl0.6Ru0.4O2 were obtained by annealing RuCl3-MIL-101 (Cr) under air for 4 h at a series of temperatures between 400 and 650 °C, denoted as Cr0.6Ru0.4O2 . As shown in the PXRD patterns  and increase with the annealing temperature  was employed to analyze the element distribution in a single nanocrystal. The resulting EDS mapping images . The Cr0.6Ru0.4O2 based electrodes were prepared by drop-casting a water/ethanol and Nafion-based ink of Cr0.6Ru0.4O2 on the glassy carbon disk (see more details in methods section). Figure 0.6Ru0.4O2 (450), Cr0.6Ru0.4O2 (500) and Cr0.6Ru0.4O2 (550) exhibit excellent initial OER activities, with onset potential of ~1.33 V vs. RHE, which represents an overpotential of ~100 mV. In addition, according to the suggested benchmark criteria33, Cr0.6Ru0.4O2 (450), Cr0.6Ru0.4O2 (500), and Cr0.6Ru0.4O2 (550) exhibited overpotentials of 175, 178 and 178 mV at the current density of 10 mA cm−2, respectively. Cr0.6Ru0.4O2 (600) and Cr0.6Ru0.4O2 (650) show slightly higher OER overpotentials , but still lower than those reported in literatures25. Note that there is little difference in the PXRD patterns for Cr0.6Ru0.4O2 electrocatalysts annealed above 500 °C, the slightly lower OER activity for Cr0.6Ru0.4O2 (600) and Cr0.6Ru0.4O2 (650) might be ascribed to the lattice strain, which was also observed on IrO234. Electrochemical impedance spectroscopy (EIS) measurement was further employed to reveal the catalytic property during OER. As shown in Fig. Rsol), charge transfer resistance (Rct), and double layer capacitance (Cdl)37. The charge transfer resistance of Cr0.6Ru0.4O2 electrocatalyst generally increases with the applied annealing temperature. The larger charge transfer resistance for Cr0.6Ru0.4O2 electrocatalyst at higher annealing temperature can also be attributed to the lattice strain effects34. For Cr0.6Ru0.4O2 (550), the charge transfer resistance is 97.2 Ω, which is much smaller than that of commercial RuO2 (>4000 Ω) tested under the same conditions  at higher voltages  has rather small Rct, with a value of 10.6 Ω at 1.47 V, corresponding to the area-specific Rct value of 0.7 Ω cm2. The durability of Cr0.6Ru0.4O2 electrocatalysts were assessed by cycling the catalysts between 1.2 and 1.6 V at a sweep rate of 100 mV s−1 in 0.5 M H2SO4 for 10,000 cycles. For Cr0.6Ru0.4O2 electrocatalyst annealed at 450 °C, the overpotential (at 10 mA cm−2) dramatically decreased from 177 mV at the first cycle to 242 mV at the 10,000th cycle  after 10,000 cycles  showed only 11 mV overpotential decrease (at 10 mA cm−2) after 10,000 cycles  was also confirmed by TEM images of Cr0.6Ru0.4O2 (550) after 10,000 cycles, where no crystallinity or morphology change was observed  were dissolved in the acidic electrolyte solution after 10,000 cycles, which results in the slight degradation of OER performance. Note that such leaching content is smaller than those of recently reported excellent OER catalysts for acidic condition27.The OER activity of Crles Fig. . The hig0.6Ru0.4O2 (550) represents the best-performance OER electrocatalyst among the Cr0.6Ru0.4O2 electrocatalysts annealed between 450 and 650 °C, with overpotential (at 10 mA cm−2) of 178 mV at the first cycle and 189 mV at the 10,000th cycle. For further comparison, the OER performance of commercial RuO2 powder with particle size of ~30 nm . The overpotential at 1 and 10 mA cm−2 of RuO2 were measured to be 240 mV and 297 mV, respectively, which are consistent with those reported in literatures  and RuO2 at the first and 10,000th cycle. The Tafel slope for RuO2 dramatically rose from 64 mV dec−1 to 88 mV dec−1 after 10,000 cycles. In contrast, the Tafel slope for Cr0.6Ru0.4O2 (550) slightly decreased from the initial value of 58 mV dec−1 to 56 mV dec−1 after 10,000 cycles. To further confirm the difference on stability of Cr0.6Ru0.4O2 (550) and RuO2 in catalytic performance, chronopotentiometry was examined under a constant current density. According to the suggested benchmark criteria in previous reports33, a current density of 10 mA cm−2 was used in the present study. Figure 0.6Ru0.4O2 (550) and RuO2. The potential for RuO2 electrocatalyst changed from 1.5 to 1.9 V in 40 min and rose sharply to 2.19 V, essentially losing all its activity. On the contrary, the Cr0.6Ru0.4O2 (550) electrocatalyst remained essentially stable throughout the 10 h chronopotentiometry test. Furthermore, the turnover frequency (TOF) of Cr0.6Ru0.4O2 (550) and RuO2 was also calculated by dividing the number of oxygen molecules generated by the number of Ru sites under an assumed 100% Faradaic efficiency  showed a TOF value of 0.15 s−1 at the overpotential of 260 mV for the first cycle and slightly decreased to 0.13 s−1 for the 10,000th cycle. However, under the same condition, the TOF of RuO2 was decreased by an order of magnitude, changing from 4.9 × 10−3 s−1 at the first cycle to 5.5 × 10−4 s−1 for the 10,000th cycle. The same TOF change trend was also observed at an overpotential of 230 mV. It should be noted that all the Ru atoms including inaccessible ones in bulk were treated as surface sites in this TOF calculation, which thus underestimated the true TOF values40. In addition, we further calculated the electrochemically active surface area (ECSA), roughness factor of Cr0.6Ru0.4O2 (550) and RuO2 electrode, and plotted the LSVs with respect to the ECSA  exhibits superior OER performance compared to RuO2 catalysts or other RuO2-based catalysts reported to date. Notably, it even outperforms the IrO2-based catalysts, which represent the state-of-the-art electrocatalyst for OER in acidic media (Table 0.6Ru0.4O2 (550) at 270 mV (229 A g−1) is also much higher than those reported in literatures. In addition, the OER performance of CrO2 powder was also measured as a reference. As expected, no OER activity was observed on CrO2 powder . Figure 3 at 450 °C, we only obtained Cr2O3 nanoparticles with high crystallinity , CrO2-RuO2 solid solution phase started to emerge after annealing. This is because RuO2 and CrO2 share the same rutile structure and have similar lattice constants, and the presence of Ru would induce the formation of RuO2–CrO2 solid solution. For Cr0.91Ru0.09O2-δ and Cr0.83Ru0.17O2-δ  with low Ru content, the major phase is still Cr2O3, which can be clearly observed from the PXRD patterns in Fig. 0.72Ru0.28O2-δ with higher Ru content, the CrO2–RuO2 solid solution turn to be the major phase, and for Cr0.67Ru0.33O2 and Cr0.6Ru0.4O2, pure phase of CrO2–RuO2 solid solution was formed. Note that, all the peaks shift slightly to the left side as the Ru content increases, which is a characteristic of RuO2–CrO2 solid solution. We further calculated the lattice parameters of Cr1-xRuxO2 with solid solution as the major or pure phase . As shown in Supplementary Figure a parameter shows the same trend as the c parameter when the Ru content increases, there is a deviation for the a parameter according to the Vegard’s law. This deviation was possibly due to the little difference of a parameter between RuO2 (a = 4.499 Å) and CrO2 (a = 4.421 Å), and/or the existence of some defects in the lattice along the a axis42. It is noteworthy that there is a pre-oxidation peak of the solid solution samples, which can be ascribed to the pre-oxidation of Cr. However, no such pre-oxidation peak was observed on Cr2O3 sample annealed at 450 °C, which can be attributed to its large crystal size  measurement of the samples to detect the residual carbon. As shown in Supplementary Figures 2–RuO2, denoted as mixed CrO2–RuO2/C. As shown in Supplementary Figure 2–RuO2/C was enhanced after the addition of carbon black, but still lower than that of pure RuO2, indicating the important synergistic effect of Cr4+ role as a participating lattice ion.We further synthesized a series of Cr–Ru oxides with different Cr ratios to investigate the Cr role on the catalytic property. By varying the mass of RuClase Fig. . However0.6Ru0.4O2 (550). As shown in Fig. d in the region between 280 and 290 eV, corresponding to the doublet peaks for Ru (IV) 3d5/2, 3d3/2 and their satellite peaks43. The primary Ru 3d5/2 and 3d3/2 peaks of RuO2 centered at 280.6 and 284.8 eV, respectively, which are consistent with literatures45. A shift to higher binding energy can be clearly observed on Cr0.6Ru0.4O2 (550) compared to RuO2, suggesting a lower electron density at the Ru sites. This can be attributed to the electron withdrawing effect of Cr (IV) in the lattice. Note that the observed C1s peaks are resulting from the carbon adhesive tape used in XPS measurement and environmental corrosion carbon. Indeed, as mentioned above, Raman characterization and thermogravimetric analysis confirmed that there is negligible carbon component in the Cr0.6Ru0.4O2 catalysts  and CrO2 in the region between 570 and 595 eV  2p3/2 of Cr0.6Ru0.4O2 (550), a shift to lower binding energy is observed compared to CrO2, implying a higher electron density at Cr sites, which confirms the withdrawing effect of Cr (IV) in Cr0.6Ru0.4O2 (550). For the other two peaks in the Cr 2p3/2 region, the smaller ones at ~575.0 eV can be assigned to Cr (III) 2p3/2, which implies the appearance of a small amount of Cr (III) sites on the outer surface of Cr0.6Ru0.4O2 (550) and CrO2 crystals48, and the larger ones at ~577.9 eV can be assigned to CrO2H, which likely resulted from the reaction between Cr (IV) and the proton from environment49. Additional XPS spectra for wide scan and Ru 3p regions are shown in Supplementary Figures We first performed X-ray photoelectron spectroscopy (XPS) to access the surface chemical state of Cr eV Fig. . The pri0.6Ru0.4O2(550), X-ray absorption spectroscopy (XAS) characterization was further employed. Figure 0.6Ru0.4O2(550). Pure Ru metal foil and RuO2 powder were also measured as reference. The shoulder near the adsorption threshold of Ru foil is corresponding to the 1s to 4d transition. For RuO2 and Cr0.6Ru0.4O2(550), this shoulder is weaker, because the increased lattice symmetry prevents the mixing of 4d and 5p orbitals. The observed transition energy of XANES (corresponding to the 1s to 5p transition) for RuO2 and Cr0.6Ru0.4O2 is higher than that for Ru. This can be attributed to the formation of Ru–O bonds, which pushes up the empty state of 5p oribials of Ru atoms50. We further analyzed the absorption energy , which is proportional to the oxidation state of transition metals52. We found that the absorption energy for Cr0.6Ru0.4O2(550) (E0 = 22129.9 eV) was similar with the value of RuO2 (E0 = 22129.5 eV), implying that the oxidation state of Ru in Cr0.6Ru0.4O2(550) is close to +4. The slightly higher absorption energy can be attributed to the electron withdrawing effect of the neighboring lattice Cr4+ ion, consistent with the XPS analysis results. Ru K-edge extended X-ray absorption fine structure (EXAFS) analysis was used to reveal the initial information on the Ru−O and Ru−Ru bonds. The corresponding Fourier transformed (FT) radial structure based on the k2-weighted EXAFS is displayed in Fig. 2 is associated with the back scattering of Ru–O in the first shell27. In contrast, the Ru–O bond length in Cr0.6Ru0.4O2(550) is slightly shortened to 1.55 Å, in line with the slightly higher absorption energy for Cr0.6Ru0.4O2(550). The peaks at 2.73 and 3.21 Å for RuO2 arise from the back scatterings of Ru−Ru in the second and third shell53. These peaks for Cr0.6Ru0.4O2(550) are assigned to the back scatterings Ru–Ru and Ru–Cr. The decreased intensity  should be ascribed to the extremely small particle size (less than 15 nm)55. Furthermore, these peaks are also shifted to the left. Clearly, the presence of Cr can profoundly alter the local electronic structures of Ru and the associated Ru-O bonding, which directly determine the OER activity. Accordingly, Cr K-edge XANES and EXAFS were also used to examine the Cr oxide state, and Cr–O bond in Cr0.6Ru0.4O2(550)  (E0 = 6006.7 eV) is higher than that of Cr metal (E0 = 5989.0 eV), but close to the value of CrO2 (E0 = 6006.8 eV). In addition, the peak in the region of pre-edge absorption is also a characteristic of the formation of Cr4+, corresponding to the 1s to 3d transition56. The slightly lower absorption energy can be attributed to the electron withdrawing effect of Cr4+ ion, in agreement with the XPS results and Ru oxidation analysis. In addition, as shown in Fig. 2 is 1.47 Å. In Cr0.6Ru0.4O2(550), the Cr–O is slightly elongated to 1.50 Å, in accordance with the EXAFS result of Ru K-edge. For comparison, we also measured the Cr K-edge XANES of Cr0.6Ru0.4O2 (450). As shown in Supplementary Figure 0.6Ru0.4O2(450) is above that of Cr0.6Ru0.4O2(550), indicating a lower symmetry environment of the Cr atoms in Cr0.6Ru0.4O2(450)56. It confirmed that the fine structure of Cr0.6Ru0.4O2(450) is different from that of Cr0.6Ru0.4O2(550), i.e., the pure phase rutile Cr–Ru oxide has not been well formed.To elucidate the atomic structure of Cr50) Fig. . The abs2–RuO2 electrocatalyst. Here we constructed a simulation model of Cr5Ru3O16, which has a composition close to the experimentally measured value. We assumed that Cr and Ru are distributed as evenly as possible in the rutile-like crystal. The simulated PXRD pattern of the relaxed structure was in good agreement with experiments. Based on the Bader charge analysis, the partial charge of Ru in bulk RuO2 was calculated to be  +1.73|e|. For comparison, the Ru cation in Cr5Ru3O16 possesses a higher positive charge of +1.92|e|. Accordingly, the partial charge on the neighboring Cr cation decreases from+1.89|e| in CrO2 to +1.84|e| in Cr5Ru3O16. Clearly, the electron transfer from Ru to Cr is consistent with the XANES results. More positively charged Ru cations lead to smaller cation radius, and the corresponding peaks in EXAFS slightly shift to the left. Moreover, the highly oxidized state of Ru implies the improved ability for the oxidation of water to oxygen, namely OER. We further plotted the density of states (DOS) to discern the nature of the electronic structures. As shown in Fig. 2. The occupation at the Fermi level decreases from 2.01 states/(eV*cell*spin) in RuO2 to 1.07 states/(eV*cell*spin) in the solid solution, indicating the stabilization of crystalline structure57. Owing to the localized nature of O p-band, its band center is known as an effective descriptor to predict intrinsic OER activity of oxides58. Considering the less electron number of Cr4+ (2e−) than that of Ru4+ (4e−), the Fermi level of solid solution is shifted downward due to the band filling effect. Correspondingly, the O p-band center moves closer to the Fermi level in Cr5Ru3O16 (−2.48 eV), compared with the value of −2.91 eV in RuO2. Clearly, the upshift O p-band suggests the enhanced activity for OER. The detailed projected DOS of Cr5Ru3O16 are displayed in Supplementary Figures 2–RuO2 structures59. The oxygen 2p (O-p) states below ~1.5 eV overlap with part of the metal d-bands. Metal t2g orbitals show a unique spread and strong peak at the edge of valence band, especially in Ru d-orbitals. Interestingly, the O-p orbital (the major component is below −2 eV) and Ru-d bands at higher energy state are well separated in RuO2  facet as the surface model, because it has been identified as the most stable from calculation of surface energy   surface was replaced by Ru, yielding a reduced RDS barrier of 1.75 eV, which is lower than that of original RuO2 surface. On the other hand, we also investigated two relatively less stable surfaces, namely (200) and (101) facets, which were observed in HR-TEM image  Fig. . It is cage Fig. . In a pr0.6Ru0.4O2 electrocatalyst with superior OER activity and stability in acidic media. Our experimental results and DFT calculations revealed the profound influence of Cr on the OER performance. The enhanced stability is related to the lower occupation at the Fermi level, while the higher activity results from the altered electronic structures. The calculated free energy diagrams for OER further demonstrates a lower energy barrier for the formation of *OOH, which is the RDS. On the other hand, Ru plays the key role to induce the formation of ruile-structured CrO2 and thus CrO2–RuO2 solid solutions because RuO2 and CrO2 share the same rutile structure and have similar lattice constants. Note that direct annealing of the MIL-101(Cr) precursor only led to an inactive product of Cr2O3. These findings and results open a route to design highly active, stable and relatively low-cost electrocatalysts for OER in acidic media. To shed light on the further optimization, we have investigated and screened a series of possible rutile-like MO2–RuO2 systems, in which M is a tetravalent cation. In light of the altered electronic structures in CrO2–RuO2, we propose that the electron withdrawing on Ru ions can facilitate water oxidation and oxygen evolution. The calculated parameters of the cells and partial charges on Ru are summarized in Supplementary Table 2–RuO2 is found to be the most positive. Interestingly, we found that MnO2 can also form solid solution with RuO2 and possess good OER performance, owing to the similar cell parameter with that of RuO2 and the highly positive partial charge on Ru (1.88 e). In contrast, we found that the reductive tetravalent cations, such as Nb and W, lead to electron accumulation on Ru. As a result, their corresponding solid solutions are expected to have lower OER activity. In addition to the formation of solid solution, we also anticipate that doping RuO2 with highly oxidizing metal ions, such as Ce4+, is another viable strategy to improve the OER activity. On the other hand, our preparation method for chromium ruthenium oxides solid solution electrocatalyst can be extended to prepare other rutile-structured electrocatalysts, such as the potentially active manganese ruthenium oxide and vanadium ruthenium oxide, or even non-Pt-group metal materials, such as chromium manganese oxide.In summary, by using Cr-based MOF, we have developed a cost effective rutile Cr2 and CrO2 were purchased from Sigma-Aldrich.All chemicals were obtained from commercial suppliers at analytical grade and used as received without further purification. The commercial RuO30. The prepared MIL-101 (Cr) was activated at 150 °C under vacuum for 12 h for future use.MIL-101(Cr) was prepared by a hydrothermal reaction following the procedure reported in our previously work3-MIL-101 (Cr) with different RuCl3 loading were prepared by mixing the desired amount of MIL-101 (Cr) and RuCl3 in THF solution. Typically, 0.2 g RuCl3 was dissolved in 30 ml tetrahydrofuran (THF) under stirring for 5 min. After that, 0.2 g MIL-101 (Cr) was slowly added into the RuCl3 solution under stirring. To ensure the uniform distribution of RuCl3 within the pores and thus the formation of homogeneous Cr–Ru oxide solid solution phase upon heating, the resulting mixture was further kept stirring at room temperature for 18 h to allow the compete loading of RuCl3 into MIL-101 (Cr) pores. After impregnation, the product was recovered by centrifugation, and washed four times with THF to remove RuCl3 remained on the outer surface of MIL-101 (Cr) particles. Finally, the resulting RuCl3-MIL-101 (Cr) was dried at 80 °C in air for 6 h. For the other RuCl3-MIL-101 (Cr) with lower RuCl3 loading, the amount of RuCl3 used was decreased to 0.15 g, 0.1 g, 0.05 g, and 0.025 g, respectively.A series of RuCl3-MIL-101 (Cr) powder was placed in muffle furnace and heated to T  at a heating rate of 5 °C/min and held for 4 h. After cooling down to room temperatures, the resulting black products were collected, and denoted as Cr1-xRuxO2 (T).Fifty microgram of RuCl1-xRuxO2 was added to 1 ml of water/ethanol  containing 15 µl Nafion aqueous solution , and dispersed by sonication for 30 min to generate a homogeneous black ink. Five microliter of the catalyst ink was drop-cast on a glassy carbon electrode (surface area: 0.07065 cm2) and dried in air at room temperature to form a thin film working electrode. For the mixed CrO2-RuO2/C ink preparation, 4 mg carbon black  was added. A three-electrode cell was employed to measure the OER electrochemical performance. The cell contained the glassy carbon working electrode, a counter electrode made of platinum wire (diameter: 0.5 nm), and a saturated Hg/Hg2SO4 reference electrode. All measurements were performed in 0.5 M H2SO4 acidic solution after purging with O2 (99.999%) for at least 30 min. The Hg/Hg2SO4 reference electrode was calibrated with a Pt wire electrode in H2-saturated 0.5 M H2SO4 solution. The potential difference between the Hg/Hg2SO4 reference electrode and reversible hydrogen electrode is 0.645 V. Cyclic voltammograms (CVs) tests were collected at a scan rate of 100 mV/s typically between 1.2 and 1.6 V. Linear sweep voltammetry (LSV) curves were recorded at a scan rate of 5 mV/s typically between 0.8 and 1.6 V. Chronopotentiometric measurements were conducted by applying constant current (10 mA cm−2) for up to 10 h. Electrochemical impedance spectroscopy (EIS) were performed at 0.75 V. The EIS results were presented in the form of Nyquist plot and fitted using ZView software with a representative equivalent electrical circuit.In a typical procedure, 4 mg of CrCDL) was determined by measuring the non-Faradaic capacitive current charging from the scan-rate dependence of CVs. The potential window of CVs was 1.21–1.31 V vs. RHE . The CDL was given by the following equation:ν is the scan rate. The slope of the plot of ic as a function of ν is equal to CDL.The ECSAs were estimated from the electrochemical double-layer capacitance of the catalytic surface. The double layer capacitance  is then calculated by dividing ECSA by 0.07065 cm2, the geometric area of the electrode.The ECSA is calculated from the double layer capacitance according to:kα (λ = 1.5418 Å) radiation at room temperature. In order to obtain high quality data for Cr0.6Ru0.4O2 (550), a very slow scan measurement was performed with a scan interval of 0.005° per step and a scan rate of 3 s per step. Structure analysis was conducted on Jade 2004. The lattice parameters were refined using GSAS software . Inductively coupled plasma-mass spectroscopy (ICP-MS) measurements were carried on NexION 300 (Perkin-Elmer). For the leaching measurements, the loading amounts of catalysts varied from 20 to 60 μg and the volume of the electrolyte was 100 ml. After 10,000 cycles, the electrolyte was concentrated to a final volume of ∼10 ml for ICP-MS analysis. The samples morphologies were examined using a field emission scanning electron microscope (SEM) . SEM specimens were prepared by depositing sample powders on carbon adhesive tape on a SEM holder. Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images were recorded on Tecnai F20 microscope, and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images were carefully recorded on Talos F200X and JEM-ARM200F. Atomic solution HAADF-STEM images were carefully recorded on JEM-ARM200F. For TEM specimen preparation, sample powders were firstly dispersed in ethanol by sonication, followed by dripping onto a carbon-coated copper grid. Nitrogen adsorption/desorption isotherms were measured on ASAP2020M apparatus at 77 K. The Brunauer−Emmett−Teller (BET) surface area was calculated over the range of relative pressures between 0.05 and 0.2. Before the measurements were performed, the samples were outgassed under vacuum at 160 °C for 12 h. X-ray photoelectron spectroscopy (XPS) spectra were recorded on the AXIS ULTRA using AlKα radiation. The X-ray absorption data (XAS) at the Ru-K edge and the Cr K edge of the samples, which were mixed with LiF to reach 50 mg, were recorded at room temperature in transmission mode using ion chambers using the BL14W1 beam line of the Shanghai Synchrotron Radiation Facility (SSRF), China. The station was operated with a Si (111) double crystal monochromator. The electron beam energy of the storage ring was 3.5 GeV and the maximum stored current was ~210 mA. The energy calibrations were performed using a Ru foil (22117 eV) or Cr foil (5989 eV). For Ru K-edge XAS, The extracted EXAFS signal, χ(k), was weighted by k2 in k-range from 3.8 to 15.6 Å−1 to obtain the magnitude. For Cr K-edge XAS, The extracted EXAFS signal, χ(k), was weighted by k3 in k-range from 3 to 12 Å−1 to obtain the magnitude.Power X-ray diffractions (PXRD) patterns of the samples were collected on a D8-Advance Bruker AXS diffractometer with Cu40. This calculation assumes 100% Faradaic efficiency:NO2 is the number of O2 turnovers, calculated using the following formula:j is the measured current density, A is the surface area of electrode, and NA is Avogadro constant (6.02 × 1023 mol−1).TOF was calculated based on the method reported in previous worksRu) is calculated using the formula: (0.4 × (20 ×10−6 g) × NA/ molecular weight of Cr0.6Ru0.4O2) for Cr0.6Ru0.4O2, and ((20 × 10−6 g) × NA/molecular weight RuO2) for RuO2, respectively.The number of Ru sites (N64. The Perdew-Burke-Ernzerhof (PBE) functional of the generalized gradient approximation (GGA)65 was employed with projector augmented wave (PAW)66 method. The valence electronic configurations were O , Ru , Cr , and H (1s). In particular, the Ueff of 3.7 eV was utilized for 3d orbital of Cr. Spin polarization was also considered. The energy cutoff for plane wave was set to 500 eV. The thresholds for electronic structure iteration and geometry relaxation were 10−5 eV and 0.03 eV/Å in force, respectively. Due to the conducting nature, the first order Methfessel-Paxton method with smearing of 0.1 eV was applied for optimization and tetrahedron method with Blöchl corrections was further used for the density of states (DOS) calculation. The lattices of RuO2 and Cr and Ru oxides solid solution were relaxed based on fixed rutile symmetry. 9 × 9 × 13 Monkhorst-Pack k-point grid was used to sample the Brillouin zone. Then four layered (110) facet was cleaved with the vacuum slab height of 20 Å. A 2 × 1 supercell containing 32 O and 16 metal atoms were studied with 5 × 5 × 1 Monkhorst-Pack k-point grid. To describe vdW interaction, empirical Grimme’s D3 correction was adopted.The DFT calculations were performed using the Vienna Ab-initio Simulation Package (VASP)The free energy of each species was calculated based on the following formula:2 was derived according to experimental standard formation energy of liquid water:The zero-point energy and entropy correction were obtained from standard vibrational calculation, whereas the free energy of O2 and other rutile-like oxides, including TiO2, VO2, CrO2, MnO2, GeO2, NbO2, MoO2, RhO2, SnO2, WO2, and PbO2. The cell sizes of the bulk models were allowed to relax in the calculations at the aforementioned level. The calculated theoretical lattice parameters are listed in Supplementary Table 2 crystals, the higher possibility the solid solution can be formed. Besides, the atomic charges on Ru atoms in these solid solution systems were calculated based on the Bader charge analysis. Here the higher positive partial charge compared with Ru in bulk RuO2 indicates that the Ru ion in solid solution would donate electrons to other metals, and accordingly its oxidizing ability is strengthened to promote OER performance. For comparison, the number of valence electron and electronegativity of various metals are labeled in Supplementary Table Moreover, we have also attempted to screen a series of potential solid solutions for further prediction, which are composed of RuOPDF version of Supplementary InformationPeer Review File


Martelella sp. strain AD-3. The corresponding protein was expressed with an N-terminal His-tag and purified by Ni2+-nitrilotriacetic acid affinity chromatography. The protein showed an overexpressed band of about 46 kDa by SDS–PAGE, and it was also proven that the enzyme contains FAD by absorption spectroscopy and HPLC analysis. The optimal activity of 3HB6H from strain AD-3 was observed in phosphate buffer (pH 8.0) at 37°C without salinity (NaCl) and metal salts. The mK values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 ± 10.1 μM and 104.1 ± 18.2 μM for 400 μM NADH and 3-hydroxybenzoate, respectively. Site-directed mutagenesis showed that residues 305, 306 and 308 are important for FAD binding. In addition, we found that Tyr221 and Gln305 of 3HB6H from strain AD-3 are involved in substrate binding.3-Hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway. We found a putative 3HB6H gene from a cluster that potentially encodes for gentisic acid degradation from a halophilic  There am-cresol . 3HB6H im-cresol . This enreaction . In the reaction . Several cepacia , Ncgl292CC 13032 , NagX frrans CJ2 , 3HB6H ftii RHA1 , and XlnIMB 9867 . Among t subunit . Recentli RHA1#2 . In addii RHA1#2 .Martelella sp. AD-3, isolated from highly saline petroleum-contaminated soil, was highly effective in degrading many PAHs with low-ring numbers, such as phenanthrene and anthracene, under broad salinities (0.1–15%) and varying pH values (6.0–10.0)  . We founsp. AD-3 . In straeviously . The 3HBEscherichia coli. The corresponding enzyme was purified and characterized. In addition, we changed residues 221, 305, 306 and 308 by site-directed mutagenesis to study their possible involvement in substrate and FAD binding.In this study, we report the expression of the gene (02545) encoding a putative 3HB6H in 3-Hydroxybenzoate (98% purity), salicylate (99% purity), 4-hydroxybenzoate (99% purity), gentisate (98% purity) and FAD (95% purity) were purchased from Aladdin , and NADH was purchased from Sangon Biotech . All other reagents and solvents used were analytical grade and the highest purity available. Luria-Bertani (LB) medium  was used for both culturing and cloning. Solid agar plates were prepared with the addition of 1.5% (w/v) agar to the LB liquid medium.Escherichia coli DH5α was used as the host for cloning experiments, while E. coli BL21(DE3) was used as the host for expressing the gene (02545). All the E. coli strains were grown in LB medium containing 50 mg/L kanamycin on a rotary incubator (200 rpm) at 37°C.1 with nucleotide BLAST and protein BLAST. These obtained sequences were compared with the sequence from strain AD-3. Conserved binding domain searches were performed using Vector NTI DNA analytical software (version 11.0). Phylogenetic analysis was performed using MEGA 7. Protein structures were predicted by I-TASSER  and HindIII (underlined) recognition sites, respectively, were designed as follows: 02545-forward (5′-CCGGAATTCATGTCAAACGTCGCAAATGA-3′) and 02545-reverse (5′-CCCAAGCTTTCAGGCCGAGACCGCGCCTT-3′). The amplified PCR product obtained was then excised with EcoRI and HindIII and ligated into pET28a(+), resulting in the recombinant plasmid designated pET28a-02545. The plasmid pET28a-02545 was subsequently transformed in E. coli BL21(DE3) to produce the protein with the His-tag located at N-terminal. E. coli BL21(DE3) carrying pET28a-02545 was cultured in LB medium containing 50 mg/L kanamycin and shaken at 37°C to an OD600 of 0.6–0.8. Then, after adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.2 mM, the cultures were incubated for 16 h at 16°C or 30°C to express the protein. The cells were harvested by centrifugation at 9,000 × g for 20 min at 4°C and resuspended in binding buffer . The cells were sonicated 40 times for 3 s at 6 s intervals with an ultrasonic disintegrator in an ice bath and then centrifuged at 10,000 × g for 20 min at 4°C to separate the soluble protein from the insoluble membrane and protein aggregates. The supernatant was used for SDS–PAGE analysis. SDS–PAGE was performed using 12.5% acrylamide for the separating gel and 3.25% acrylamide for the stacking gel.The gene (02545) was amplified from the genomic DNA of strain AD-3 using pfu DNA polymerase . The primers, which are the 600 of 5.0 in 50 mM phosphate buffer (pH 8.0) containing 200 μM 3-hydroxybenzoate at 200 rpm and 30°C. During the course of the conversion, the concentrations of 3-hydroxybenzoate and gentisate were monitored by High Performance Liquid Chromatography (HPLC) and Liquid Chromatograph-Mass Spectrometer (LC-MS) systems . For HPLC analysis, the sample of direct sampling was centrifuged at 10,000 × g for 2 min. And then the supernatant was filtered through a 0.22 μm millipore filter to prepare the samples. 10 μL of each sample was injected onto an Eclipse XDB-C18 column  and run at 30°C with 50% methanol and 50% water (containing 1‰ formic acid) as the eluent at a flow rate of 0.8 mL/min. The concentrations of 3-hydroxybenzoate and gentisate were detected by UV spectroscopy at 235 nm and 320 nm. For LC-MS analysis, the conditions of liquid chromatography were the same as those of HPLC, and the mass spectrum adopted a negative ion mode.Small-scale resting cell assays were performed as described previously  using an6-tagged 3HB6H from strain AD-3 was eluted from the column using elution buffer . All the purification steps were carried out at 4°C.The cell cultures were induced for 16 h at 30°C and then disrupted by ultrasonic wave. After centrifugation, the supernatant was filtered through a 0.22 μm filter, and the resultant filtrate was applied to a 5 mL column of Ni-NTA agarose  that had been equilibrated with the binding buffer. After a wash with 30 mL of washing buffer , His360 = 3.50 mM-1⋅cm-1  were completed while maintaining a constant enzyme concentration in 50 mM potassium phosphate buffer (pH 8.0) at 30°C. Kinetic parameters were determined from Lineweaver-Burk plots.A series of 3-hydroxybenzoate solutions ranging from 50 to 1000 μM was prepared to determine the EcoRI (underline) and HindIII (underline). The recombinant pET28a that contained the mutant genes was transformed into E. coli BL21(DE3) followed by overexpression and purification. The strain growth, protein purification and enzyme assay methods are described above.Site-directed mutagenesis was performed by using a recombinant PCR method. The primers are listed in Supplementary Table Pseudomonas alcaligenes NCIMB 9867 . The complete sequence alignment is shown in Supplementary Figure Putative 3HB6H from strain AD-3 was found to belong to a family of group A flavoprotein monooxygenases that includes salicylate 1-hydroxylases, 3-hydroxybenzoate 4-hydroxylases and so on. . It was IMB 9867  and onlytii RHA1 . A phyloE. coli BL21(DE3) containing the plasmid pET28-02545 was induced for 16 h in LB medium supplemented with 0.2 mM IPTG at 16°C and 30°C, respectively. The SDS–PAGE revealed an overexpressed band of about 46 kDa and showed the amount of soluble protein which induced at 30°C was more than that which induced at 16°C in same conditions . To determine the protein encoded by the gene (02545) is a 3-hydroxybenzoate 6-hydroxylase, we used resting cell reaction to detect the conversion of hydroxybenzoate at 30°C by HPLC. The Figures 2B,C clearly showed that the concentration of 3-hydroxybenzoate decreased and the concentration of gentisate increased. The mass spectrometry data of LC-MS confirmed that the substracte was 3-hydroxybenzoate and the product was gentisate . Thus, the protein encoded by the gene (02545) is a 3-hydroxybenzoate 6-hydroxylase.The gene (02545) was cloned into the expression vector pET28a. Figure 3A). And the Native-PAGE showed a band of 90 kDa. Therefore, the 3HB6H from strain AD-3 might be dimeric protein . The A274/A452 ratio of the FAD-saturated protein preparation was 11.5. The FAD concentration in the purified 3HB6H (0.29 mM) was 0.30 mM, and FAD/enzyme ratio was 1.02 ± 0.05 by HPLC analysis. The result of identifying protein-bound lipids showed that the 3HB6H expressed in E. coil contained phosphatidylglycerol, while phosphatidylethanolamine was not detected  over the course of time in reaction mixture containing 200 μM NADH and 200 μM 3-hydroxybenzoate . The activity was measured 60 s after the addition of 3HB to reactions with different pH buffers. Citric acid-sodium citrate, phosphate, Tris–HCl, and sodium carbonate-sodium bicarbonate buffers were used for incubation at pH 4.0–6.0, 6.0–8.0, 8.0–9.0, and 9.0–11.0 with identical concentration (50 mM), respectively. 3HB6H showed maximal stability in phosphate buffer (pH 8.0). However, Tris–HCl (pH 8.0) obviously inhibited the activity of AZF01_02545 . The effect of metal salts at 0.5 mM  was examined. Ca2+ and Mn2+ had no effect on enzyme activity. Mg2+, Fe3+, Cu2+, Zn2+, and Co2+ reduced enzyme activity. The addition of Cu2+ reduced enzyme activity to 16.8%, and even the addition of Zn2+ reduced enzyme activity to 22.6% . To assess temperature stability, 3HB6H was incubated in phosphate buffer  for 60 min at temperatures ranging from 16 to 50°C for assay activity. The enzyme activity was maintained at 16–37°C, and quickly lost at higher temperatures. The enzyme precipitated initially at 42°C and precipitated completely at 50°C . The influence of reaction temperature on enzyme activity was also investigated at the temperature range from 16 to 50°C in phosphate buffer (pH 8.0). The maximal enzyme activity was observed at 37°C, and the specific activity was 25.2 U/mg .The effect of salinity on 3HB6H activity was measured in phosphate buffer (pH 8.0) at 30°C, with salinity ranging from 0 to 85.56 mM. With the increase in salinity, enzyme activity clearly decreased. The activity reached to 3% in 85.56 mM salinity . Therefore, the maximum activity is 25.2 U/mg in optimal conditions which is phosphate buffer (pH 8.0) at 37°C without salinity (NaCl) and metal salts.Protein 3HB6H was purified by using Ni-NTA, and the yellow protein was obtained. SDS–PAGE showed an overexpressed band of about 46 kDa, which is 3HB6H . At a constant level of 3-hydroxybenzoate (0.4 mM) and various amounts of NADH, the protein exhibited an apparent mK of 104.1 ± 18.2 μM and catk of 7.8 ± 0.4 s-1 for NADH .The purified 3HB6H displayed typical Michaelis-Menten kinetics, and Lineweaver-Burk plots of enzyme activity yielded an apparent Figure 1B). The GxGxGG moiety (Gly15 Gly17 Gly19 Gly20 in 3HB6H from strain AD-3) is a conserved motif at the N-terminal region of NAD(P)H-dependent and FAD-containing oxidoreductases. This moiety is important for binding the ADP moiety of FAD . In addition, we substituted a site (Tyr221) that was different from most salicylate hydroxylases in the same site (Phe) and obtained Y221F. The mutants were then purified; the enzyme activity and the amount of FAD binding of these mutant proteins compared to the wild-type protein are shown in Martelella strain.3HB6H plays an important role in the gentisate pathway of degradation 3HB in many bacteria. Previous reports were all found in non-halophilic organisms. This is the first report of a 3HB6H found in a halophilic Figure 1B). Full-wavelength scanning showed a typical characteristic of flavoproteins . The A274/A452 ratio of the FAD-saturated protein preparation was 11.5, and each 3HB6H subunit contained one FAD. This result showed that the 3HB6H from strain AD-3 is similar to Rj3HB6H  and had no enzyme activity when salicylate and 4-hydroxybenzoate were used as substrates, similar to other 3-hydroxybenzoate 6-hydroxylases (data not shown) (Rhodococcus jostii RHA1), XlnD , and NagX   . The molivorans) .3+, Cu2+, Zn2+, Co2+, and Ba2+ reduced the activity of all of the enzymes  in phosphate buffer (pH 8.0) at 37°C. This enzyme has relatively high activity after incubating at 16–37°C for 1 h, but the activity is 7.21 U/mg when the system environment is at 16°C. And there were high reaction speed at 42°C to 50°C, but the enzyme lost activity rapidly. Therefore, 37°C is the optimal temperature which guarantee no loss of enzyme activity and ensure to keep high activity.Comparing the effect of metal salts with XlnD, Ncgl 2923, and MhbM, Fe enzymes . Ca2+ anMartelella strain AD-3, which is a moderate halophilic bacterium, can degrade 3-hydroxybenzoic acid in high salinity environments in which NaCl is the main source of salt. However, the activity of the purified 3HB6H decreased significantly as the amount of salinity increased. This phenomenon is similar to that observed with other flavoproteins; Cl- has a strong inhibitory effect on flavoprotein monooxygenases  than in Tris–HCl (pH 8.0). To explore whether 3HB6H catalysed 3-hydroxybenzoate under high salinity conditions (NaCl), we conducted a salinity experiment. ygenases . In Rhod or both .Rhodococcus jostii RHA1 (Table 1). The FAD-binding ability of Y221F was similar to that of the wild-type enzyme. The mK of Y221F for 3HB was the highest among wild-type and mutants, and showed 2.3 times that of the wild-type, however, its enzymatic activity was less than that of the wild-type. Therefore, it was concluded that Y221F may be associated with substrate binding. In Rj3HB6H, Tyr217 is the important residue for substrate binding, which results in a hydrogen-bond interaction with the carboxylate group of 3HB. When substituting Tyr217, there is a substantial decrease or complete abolishment of substrate binding (mK of Q305P to 3HB was greater than that of the wild-type, and the binding amount of FAD was smaller than that of the wild-type. Therefore, Q305 was related to the binding of the substrate and FAD. This result validates previous findings in Rj3HB6H. When this site was changed to Glu, the enzyme lost activity. This site had a primary role in catalysis, possibly facilitating deprotonation of the substrate phenol (Rj3HB6H in which the A304 was related to bind FAD (Based on the reported structure of 3HB6H in tii RHA1  and the  binding . Among te phenol . When webind FAD .HT and CC conceived and designed the experiments. XC performed the experiments. KL and YX analyzed the data. PX and YL contributed reagents, materials, and analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from  The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry and its autoinhibition is released by PI4P binding. Here authors show cryo-EM structures of Drs2p-Cdc50p in apo and PI4P-activated form which reveal the structural changes upon PI4P binding. Drs2p belongs to a large family of Type IV P-type ATPases (P4 ATPases)2. These enzymes translocate specific phospholipids from the extracellular side of the plasma membrane or from the lumenal side of internal organelles to the cytosolic side, creating and maintaining lipid compositional asymmetry in eukaryotic cell membranes. Drs2p has an architecture similar to that of several cation-transporting P-type ATPases, having an actuator (A) domain, a nucleotide-binding (N) domain, a phosphorylation (P) domain, and a transmembrane domain (TMD) of 10 transmembrane helixes (TMs)3. The N-terminal and C-terminal peptides of Drs2p inhibit the enzyme activity5. Cdc50p functions as a chaperone; its association with Drs2p is required for stability and ER export7, but Cdc50p may also contribute to Drs2p activity via an unknown mechanism8.The yeast lipid flippase Drs2p-Cdc50p is primarily found in the trans-Golgi network and is involved in protein trafficking between that network, the plasma membrane, and endosomes9. Drs2p-Cdc50p is activated by the binding of phosphatidylinositol 4-phosphate (PI4P), the Arf guanine nucleotide exchange factor Gea2p, and the Arf-related small-GTP-binding protein Arl1p to the C-terminal region of Drs2p10. Phosphatidylserine appears to be the primary substrate of Drs2p-Cdc50p13, although phosphatidylethanolamine can also be transported to some extent14. We sought to understand how the enzyme complex inhibits itself and how PI4P activates the flippase activity by determining the cryo-EM structures of the Drs2p-Cdc50p complex in the autoinhibited apo form and in the PI4P-activated form. Because we use the intact complex rather than a C-terminal truncated form, the conformational changes we observe in Drs2p upon PI4P incubation may be a more faithful representation of the activation events that occur in vivo.Drs2p-Cdc50p alone is in an autoinhibited inactive formS. cerevisiae Drs2p and purified the complex from baker’s yeast using an affinity column followed by size-exclusion chromatography  are disordered in the cryo-EM map. The disordered extreme C-terminal region may not to be functionally important and does not participate in the autoinhibition of the enzyme, because removal of this peptide by truncation does not stimulate the Drs2p activity. In other words, the truncated Drs2p remains in the inhibited state as long as the first 60% of the ordered C-terminal peptide is present5.The yeast Drs2p is a large protein with 1355 residues Fig. . As expe2+-ATPase (SERCA), which is a P2 ATPase whose structure has been extensively characterized in the literature16. We compared the apo Drs2p structure with those of the SERCA ATPase , and found that our Drs2p structure differed from all four major conformations  of SERCA16  are also regulated by their C-terminal peptides18. In fact, the structure of the human PMCA1 in complex with its obligatory subunit neuroplastin was recently reported, but the inhibitory C-terminus was not visualized in the 4.1-Å resolution cryo-EM 3D map19.The catalytic cycle of a P-type ATPase requires ATP binding in the nucleotide-binding pocket in the N domain, followed by phosphorylation of an Asp in the P domain, leading to a rotation of the A domain that subsequently modulates the conformation of TMD and thus the cation translocation activityail Fig. . The C-tail Fig. , so that5. In our structure, most of the N-terminal peptide is disordered except for the 17-residue region (Phe-196 to Thr-212) before reaching TM1. The ordered N-terminal peptide extends over both the P and A domains 22 to identify a putative substrate-transporting in Drs2p  have been demonstrated to determine the specificity for phosphatidylserine transport10. We obtained the activated complex by incubating the purified sample with 26 μM PI4P for 15 h at 4 °C, resulting in a cryo-EM structure determined at 3.3 Å resolution  theion Fig.  becomes ion Fig. .Fig. 5ThAs discussed above, the Drs2p C-tail inhibits the Drs2p activity by interacting extensively with the A, N, and P domains of Drs2p and occupying the ATP-binding pocket. In the structure of the active Drs2p-Cdc50p, the entire Drs2p C-tail becomes disordered, thus vacating the ATP-binding pocket of Drs2p previously occupied by Phe-1275 and Phe-1277 Fig. . The A, The ordered C-terminal inhibitory peptide is connected to the last transmembrane helix, TM10, by a short α-helix see Fig. . In the 9, we suggest that this is the PI4P binding pocket and that PI4P binding at this site allosterically regulates the flippase activity of the enzyme complex23. Because the substrate PS is also negatively charged, we do not exclude the possibility that it may also bind in this pocket.As we mentioned above, PI4P, which activates the enzyme, is absent in the cryo-EM map, suggesting that after it completes the activation of the enzyme, PI4P diffuses away from the binding pocket. We observed a highly positively charged pocket close to and just above the helix switch, which is lined by Trp-1223, Lys-1224, Lys-1227, Arg-1228, Tyr-1235, and His-1236 Fig. . Becausedrs2Δ strain which has a cold-sensitive growth-defect phenotype; this deficiency can be complemented by a plasmid carrying the wild-type drs2 gene. We introduced five individual mutations in the putative PI4P binding site  and performed complementation assays on drs2Δ cells. We found that only Y1235A and H1236A significantly disrupted Drs2 function in vivo, as these mutants were unable to rescue the drs2Δ yeast growth at 20 °C than those plasmids carrying the WT drs2  , 1.5% n-dodecyl β-D-maltoside (DDM), 0.15% cholesteryl hemisuccinate Tris salt (CHS), 0.5 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM EDTA, and 1 mM PMSF. After incubation for 30 min at 4 °C, the mixture was centrifuged for 30 min at 120,000 × g, and the clarified supernatant was loaded into a home-packed anti-FLAG (M2) affinity gel column at 4 °C. The column was then washed three times in buffer B containing 0.025% DDM, 0.0025% CHS, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM MnCl2. Finally, the Drs2p-Cdc50p complex was eluted with three column volumes of buffer B containing 0.15 mg/mL 3×FLAG peptide, concentrated to 500 μL with a 100-kDa cutoff centricon. The sample was further purified in a Superose 6 10/300 gel filtration column in buffer C containing 0.01% lauryl maltose-neopentyl glycol (LMNG), 0.001% CHS, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM MgCl2, and 1 mM MnCl2. For the active Drs2p-Cdc50p, the purified sample in Buffer C was incubated with 26 μM PI4P for 15 h at 4 °C. The final sample was assessed by SDS-PAGE gel and the identities of the protein bands were confirmed by tryptic digestion followed by mass spectrometry.The C-terminal triple FLAG-tagged Drs2 construct was generated using a PCR-based genomic epitope-tagging method on the yeast W303-1a  and were flash-frozen in liquid ethane using an FEI Vitrobot Mark IV. The micrographs were screened in a 200 kV FEI Arctica electron microscope. A small data set of 200 micrographs collected in this microscope on an FEI Falcon III direct detector led to some 120,000 raw particles. After 2D classification, about 70,000 particles were selected for 3D reconstruction, which resulted in a 6.2-Å preliminary 3D map. A full data set was collected during a 4-day session in a 300 kV FEI Titan Krios electron microscope operated at a nominal magnification of ×130,000 and a pixel size of 1.029 Å per pixel with defocus values from −1.0 to −2.0 μm. The dose rate was 2 electrons per Å25. Contrast transfer function parameters of each aligned micrograph were calculated using CTFFIND 4.126. All the remaining steps were performed using RELION 327. For apo form, 2970 raw movie micrographs were collected. Templates for automatic picking were generated from a 2D average of about 1000 manually picked particles. A total of 1,040,625 particles were picked automatically. 2D classification was then performed and particles in the classes with features unrecognizable by visual inspection were removed. A total of 1,001,980 particles was used for further 3D classification, and 635,300 particles were selected for further 3D refinement and postprocessing, resulting in the 2.8-Å 3D density map. The resolution of the map was estimated by the gold-standard Fourier shell correlation at a correlation cutoff value of 0.143. For active form, 4717 raw movie micrographs were collected. A total of 1,126,540 particles were picked automatically. After 2D classification, a total of 897,023 particles was used for 3D classification, and 498,745 particles were selected for further 3D refinement and postprocessing, resulting in the 3.3-Å 3D density map.The movie micrographs collected from the Titan Krios were motion-corrected using the program MotionCorr 2.0https://swissmodel.expasy.org). The model was split into four domains: TM, A, N, and P domains, which were then fitted into the EM density independently in Chimera28. Initial model of Cdc50p was first automatically built using the model_to_map in the PHENIX program29 and then was manually built in the program COOT30. The complete Drs2p-Cdc50p model was refined by real-space refinement in the PHENIX program and subsequently adjusted manually in COOT. Active Drs2p-Cdc50p model was then built into the 3.3-Å 3D density map basing on the apo model. Notably, the A and N domains of Drs2p were only built by rigid-body fitting because of their low local resolution at 4–6 Å. Finally, the atomic model was validated using MolProbity31. To avoid overfitting, the 3D maps of the final map and the two half-maps  were correlated with the refined model to produce three FSC curves: Model vs. final map, FSCwork , and FSCfree . Structural figures were prepared in Chimera28 and PyMOL (https://pymol.org/2/).We first built the apo Drs2p-Cdc50p model into the 2.8-Å 3D density map. The initial model of Drs2p was generated based on PDB ID 5YLV using online SWISSMODEL (2 at 37 °C for 1 h. The sample without PI4P incubation was used as control. Released phosphate was measured colorimetrically using Malachite Green Phosphate Assay Kit from Sigma.Purified Drs2p-Cdc50p was first incubated with 26 μM PI4P for 15 h at 4 °C, and then assayed for ATPase hydrolysis activity in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% LMNG, 0.001% CHS, 10 mM MgCldrs2Δ strain and the complement plasmid carrying drs2 gene were gifts from Prof. Todd Graham in Vanderbilt University. Drs2 mutants were generated by QuikChange mutagenesis protocol with Pfu DNA polymerase and desired mutation primers (Supplementary Table drs2Δ yeast transformants carrying either wide-type drs2 plasmid or mutations, the strains firstly grew to the same OD in + G418 YPD medium at 30 °C. Then 7 μL of 1:10 serial dilutions of the cells were spotted onto + G418 YPD plates, incubated at 20 or 30 °C for 2 days, and examined for growth.Yeast Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting SummarySource Data


Ferula lutea flowers. Total phenolics and flavonoids of the n-butanol and ethyl acetate extracts were quantified . Two diastereoisomers were isolated and identified using spectroscopic techniques . The extracts and diastereoisomers were tested for antioxidant, antiacetylcholinesterase, antimicrobial, antidiabectic, cytotoxic (leukemia cell line) activities and allelopathic potentialities. The strongest antioxidant activity was obtained for the ethyl acetate extract (IC50 = 12.8 ± 1.29 µg/mL). The two extracts exhibited high antidiabetic activity (54.1 and 52.1% at 40 µg/mL).The present work describes the phytochemical study on  Plants still serve as a far reaching source of innovative and original compositions on the structural level. These new compounds serve as a clue for the discovery of new medicines, herbal and practical foods. There is an escalating interest in naturally occurring antioxidants to replace synthetic counterparts used for food conservation, flavoring, and cosmetics, as well as in health care. Ferula (family Apiaceae) comprises 170 species + at m/z 328 indicating a common molecular formula of C19H20O4. The structure of 1a and 1b  and δH 7.57  as AB-type signals, attributable to H-3 and H-4 in 1a, respectively. A second AB-type spin system was also observed at δH 6.20  and δH 7.56 , corresponding to H-3 and H-4 in 1b, respectively. The  deltoin  19]. Th. Th1H-NMH 6.69  and δH 7.19  and at δH 6.76  and δH 7.14  corresponding to the aromatic protons H-8 and H-5, respectively. The observation in the same spectrum of a triplet at δH 5.02  and a multiplet at δH 5.12  a multiplet at δH 3.10–3.30  suggested the presence of the dihydrofuranic moiety in 1a and 1b [1a and 1b. The two methyl groups attached at C-4' in both 1a and 1b were identified according to the two pair of singlets at δH 1.58  and 1.59  and at δH 1.37  and 1.39 , respectively. The other signals at δH 5.95 , at δH 1.63 , at δH 1.87 , at δH 6.09 , at δH 1.63 , at δH 1.85  coincide with the spectral data of an angeloyl moiety in 1a and 1b with two different configurations  to the literature data [Z) configuration of the double bond in the angeloyl fragment in 1a was evidenced from the NOESY spectrum showing the dipolar interaction of the ethylenic proton H-3''  with both methyl groups (CH3)-4''  and (CH3)-5'' , whereas, the (E) configuration of the same double bond in the angeloyl moiety in 1b was proved by the unique nOe between H-3" , and H-5"  revealed from the same NOESY spectrum.The stereochemical proposal for carbon C-2' in ure data . On the 1a and 1b are grouped in 1a was never previously isolated from any Ferula species, but it was previously isolated from Seseli peucedanoides (roots) and Heracleum candolleanum (seeds or roots), while compound 1b was isolated from Peucedanum japonicum (roots), Seseli resinosum (roots) and Ferula lutea (roots). Compounds 1a and 1b were identified for the first time in the F. lutea flowers. The various plants and organs containing compounds F. lutea flowers are shown in n-butanol extract had the highest quantity (52.3 ± 0.4 mg GAE/g of dry weight) followed by the ethyl acetate extract (40.7 ± 0.2 mg GAE/g of dry weight). The flavonoids content of the n-butanol extract was the highest (14.7 mg QE/g dry weight) while the ethyl acetate extract had 12.4 ± 0.1 mg QE/g dry weight. The phenolics and flavonoids content of F. lutea has not been reported in the literature. The study of other species and organs  showed that the total phenolics were 94.8 ± 5.9 mg GAE/g of dry weight and total flavonoids were 90.9 ± 6.9 mg QE/g dry weight [The amount of total phenolics and flavonoids content in the extracts of y weight .50 = 12.8 ± 1.3 µg/mL) followed by the n-butanol extract (IC50 = 26.0 ± 1.8 µg/mL). The two extracts have good antioxidant activity compared to the positive control, L-ascorbic acid . The ABTS assay confirmed the DPPH assay results, with the ethyl acetate showing an IC50 = 184.0 ± 7.0 µg/mL and therefore better antioxidant activity than the n-butanol extract (IC50 = 300.0 ± 5.0 µg/mL). The antioxidant activity (DPPH and ABTS) of F. lutea extracts has not been cited in the literature before. For this genus, only F. gummosa Boiss roots [50 = 579.6 ± 19.4 µg/mL.The antioxidant activity of the extracts assayed using two methods (DPPH and ABTS) is presented in F. lutea flower extracts was tested  exhibited more interesting antimicrobial activities than the ethyl acetate extract (MIC and MBC: 10.00 mg/mL), being especially active against Staphylococcus aureus with a MBC and MIC value of 1.25 mg/mL.The antimicrobial activities of Candida yeasts, the ethyl acetate extract (MIC and MBC: 0.30–1.25 mg/mL) exhibited more interesting activities than the n-butanol extract (MIC and MBC: 10.00 mg/mL). The ethyl acetate extract was especially active against C. parapsilosis, C. albicans and C. kreseui, with a MFC value of 0.625 mg/mL and MIC value of 0.3 mg/mL; C. glabrata was found to be the most resistant species, with MIC of 0.625 mg/mL and MFC of 1.25 mg/mL, compared to the positive control, thymol . No data on the antimicrobial activity of F. lutea or other Ferula spp. extracts is available.Concerning F. lutea extracts against α-amylase is shown in n-butanol extracts showed very good α-amylase inhibitory activity. At 20 μg/mL of extracts, the inhibitory activity was 54.1% and 52.1% for the ethyl acetate and n-butanol extracts, respectively. The mixture of compounds 1a and 1b was poorly active (IC50 > 100 µg/mL). Acarbose is less active than these two extracts. To our knowledge, no previously study on the antidiabetic activity of any Ferula species has been reported.The inhibitory activity of n-butanol extract, but 87% for the mixture of compounds 1a and 1b, then compounds a and 1b . Indeed,a plants . Relativn-butanol extracts using the cancer cell Line, K562 (leukemia cell line) are given in n-butanol extract was superior to that of all samples tested with an IC50 = 40 µg/mL, while the ethyl acetate extract and compounds 1a and 1b were poorly active (IC50 > 100 µg/mL). The value of the cytotoxic activity of the n-butanol extract and is good, but not very high compared with the standard substance (doxorubicin).This is the first work citing the cytotoxic activity of F. lutea or other Ferula spp.The results of the anticancer test on the isolated compounds, ethyl acetate and 1H- (300 MHz), 13C- (75 MHz) and 2D NMR spectra of compounds 1a and 1b were recorded in CDCl3 with a Bruker NMR-300 spectrometer. The residual solvent resonances were used as internal references. Chemical shifts are expressed in ppm and coupling constants are given in Hertz.F. lutea flowers were collected in the region of Béja (Tunisia) on April 2010 and identified by Professor Féthia Harzallah Skhiri, at the Laboratory of Genetic Biodiversity and Valorisation of Bioresources of the Higher Institute of Biotechnology of Monastir, Tunisia. A voucher specimen (F.L.F-10) was deposited in the same laboratory.F. lutea (5.8 kg) were macerated at room temperature with MeOH–H2O  at room temperature for 7 days and then concentrated by rotary evaporation leaving an oil layer (5.8 g) and an aqueous residue (only the organic solvent was evaporated at the low temperature used ‒30 °C) which was partitioned successively with ethyl acetate and n-butanol extracts, yielding, after evaporation of the solvents, the corresponding ethyl acetate (56 g) and n-butanol (220 g) extracts. From the all extracts, only the oil layer (5 g) was subjected to silica gel column chromatography eluting successively with pentane–ethyl acetate (95:5 to 100% ethyl acetate) to afford 14 fractions. The ninth subfraction (2.4 g) was chromatographed on silica gel eluting with pentane–ethyl acetate (7:3) to afford a mixture of compounds 1a and 1b (2 g) that was difficult to separate. The 1H- and 13C-NMR spectral data of two diastereoisomers compounds  are listed in The fresh flowers of et al., [2CO3) solution (750 µL) was added to the mixture and then gently mixed. After standing at 25 °C for 90 min, the absorbance was read at 725 nm using an UV–Visible spectrophotometer. A standard curve of gallic acid was used. Total phenolic content of plant parts was expressed as mg gallic acid equivalents per gram of dry weight (mg GAE/g DW) through the calibration curve with gallic acid. The calibration curve range was 0–250 μg/mL (R2 = 0.99).The amount of total phenolic compounds was determined according to the method of Velioglu et al.,  which us3 method [3·6H2O (2 g in 100 mL methanol). The mixture was vigorously shaken, and absorbance at 367 nm was read after 10 min of incubation. Total flavonoid content was expressed as mg quercetin/g dry weight (mg QE/g DW), through the calibration curve of quercetin. The calibration curve range was 0–50 μg/mL (R2 = 0.99). The AlCl3 method  was used.radical was calculated using the following equation:Diluted methanol solutions of the samples at different concentrations (1 mL each) were mixed with 1 mL of a freshly prepared (80 µg/mL) methanol solution of DPPH. The resulted solution was incubated at 37 °C for 30 min after what the absorbance of the solution was measured at 517 nm [50 was calculated using the linear relation between the compound concentration and the probability of the percentage of DPPH inhibition.The IC+. solution was added to 1 mL of methanolic solutions of extract at different concentrations and the absorbance at 734 nm was measured 10 min after the initial mixing, using methanol as the blank. The percentage of inhibition was calculated by the equation:control is the absorbance of the control reaction containing all reagents except the tested sample, and Asample is the absorbance of the test compound. Tests were carried out in triplicate. Ascorbic acid was used as the positive control. The IC50 was calculated using linear relation between the compound concentration and probability of the percentage of ABTS inhibition.ABTS was dissolved in water to a 7 mM concentration and the ABTS radical cation was produced by adding potassium persulfate to a final concentration of 2.45 mM . The radcontrol is the absorbance of the control reaction containing all reagents except the tested sample, and Asample is the absorbance of the test compounds. Tests were carried out in triplicate and a blank with Tris-HCl buffer instead of enzyme solution was used.Briefly, 50 mM Tris–HCl buffer , the extract (50 µL) and acetylcholinesterase solution (10 µL) containing 0.26 U/mL were mixed in a microwell plate and left to incubate for 15 min. Subsequently, a solution of AChI  and 3 mM DTNB (140 µL) were added. The absorbance was read at 405 nm when the reaction reached equilibrium . Eserineet al. [2. The resulting blue formazan product can be measured spectrophotometrically. The MTT colorimetric assay was performed in 96-well plates. Adherent human chronic myelogenous leukaemia K562 cells were seeded in a 96-well plate at a concentration of 104 cells/well and incubated at 37 °C overnight in a 5% CO2 enriched atmosphere. Cells in exponential growth phase were incubated at 37 °C for 72 h with each tested compound at concentrations ranging from 3.12 to 400 μg/mL. After that, the medium was removed and cells were treated with MTT solution  at 37 °C for 2 h. MTT solution was then discarded and DMSO (500 μL) was added to dissolve insoluble formazan crystals. Optical density was measured at 540 nm. Each extract concentration was tested in triplicate. Doxorubicin was used as positive control.Cytotoxic activity of extracts against the human chronic myelogenous leukaemia cell line K562 was estimated by the 3--2,5-diphenyltetrazolium bromide (MTT) assay of Khlifi et al. . The cel1a and 1b (diluted to 1% in the hexane) was achieved by coupled GC/MS analysis, on a system composed of a gas  coupled to a quadrupolar mass spectrometer (HP-MSD 5972 TO) operated in electron impact mode. The column used was an HP polar INNOWAX column of 30 m, 0.25 mm, and 0.25 μm film thickness. Its stationary phase was constituted of polyethylene glycol.The mass spectra of compounds 2. The tubes containing substrate solution were boiled for 5 min. After 5 min of preincubation at 37 °C, extract dissolved in DMSO (0.2 mL) was added to the tube containing the substrate solution. A volume of porcine pancreatic amylase in Tris-HCl buffer (0.1 mL) was added and then incubated at 37 °C for 10 min and the reaction was stopped by adding 50% acetic acid (0.5 mL). The reaction mixture was centrifuged at 3,000 rpm for 5 min at 4 °C and the absorbance was measured at 595 nm.Porcine pancreatic α-amylase inhibitory activity was determined using a literature method . Starch Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212; Gram-negative bacteria: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 and four Candida yeasts: Candida albicans ATCC 90028, C. glabrata ATCC 90030, C. kreusei ATCC 6258 and C. parapsilosis ATCC 22019).The test microorganisms included the following Gram-positive bacteria: et al. [4 µg/mL. The MIC of each sample was defined as the lowest concentration of oil that inhibited either the bacterial or candidal growth, after incubation at 37 °C for 18 to 24 h. The MBC and the MFC were determined by subculture on blood agar at 37 °C for 18 to 24 h. Thymol and gentamicin or thymol and amphotericin B were used as positive controls against the bacterial or candidal strains, respectively. The highest activities for each sample are presented in The MIC values for the antibacterial and anticandidal screening were determined with the broth dilution method (microdilution using 96-well microplates) following the procedure described by Cintia et al. . Brieflyn-butanol (BE) extracts and the mixture (1a + 1b) (C1) were again dissolved in MeOH to compare their phytotoxic effects. Five mL of each organic fraction at 6,000 ppm and mixture (1a + 1b) at 1,000 ppm were placed in a Petri dish lined with a sheet of Whatman No. 1 filter paper and evaporated to dryness for 24 h at 24 °C. After evaporation, distilled water (5 mL) was pipetted onto the filter paper. Methanol and distilled water were both were used as negative control. Seeds of lettuce were surface sterilized by immersing in 0.525 mg/mL sodium hypochlorite for 15 min, and then were rinsed four times with deionised water, imbibed in deionised water at 22 °C for 4 h, and carefully blotted using a folded paper towel. Lettuce has been used as tested plant because it is very sensitive to chemicals at low concentration, although it sometimes overestimates the actual allelopathy [The two samples concentrated from the ethyl acetate (EAE) and elopathy . Thirty elopathy . Data weF. lutea flowers were investigated for their chemical composition. Total phenols and flavonoids contents of extracts were determined and two diastereoisomers were isolated and identified. To the best of our knowledge, we report the first study on the antiacetylcholinesterase, antidiabectic, antimicrobial, allelopathic and cytotoxic activities of F. lutea flower constituents. We can conclude that the ethyl acetate extract has the highest antioxidant activity, the n-butanol one possesses an interesting cytotoxicity and finally both extracts have good antidiabetic activity. Nevertheless, it is interesting to continue the fractionation work to isolate the molecules responsible for the antioxidant, antidiabetic and cytotoxic activities. Compounds 1a and 1b were isolated as a mixture and their activities were evaluated for the first time.


Rotavirus is a leading cause of severe diarrheal disease, and one of the common causes of death in children aged under five years old. The dominant epidemic strains may change in different years in the same area. In order to provide evidence for rotavirus epidemic control and inform vaccine development, we analyzed epidemiological patterns and genetic characteristics of rotavirus in Beijing during 2011–2016.Stool specimens of outpatient children under five years old were collected from three children’s hospitals on a weekly basis. Group A rotavirus antigens were detected using enzyme-linked immunosorbent assay (ELISA) kit. The partial VP4 genes and VP7 genes of rotavirus were both amplified and sequenced. Genotyping and phylogenetic analyses were performed. Logistic regression and Chi-square tests were performed to determine differences across age groups, districts and years in rotavirus prevalence and genotype distribution.A total of 3668 stool specimens from children with acute diarrhea identified through hospital-based surveillance were collected from 2011 to 2016 in Beijing. A total of 762 (20.8%) specimens tested positive for rotavirus. The rotavirus-positive rate was highest among the 1–2 years old age group . November, December and January were the highest rotavirus-positive rate months each year. G9 was the most common G genotype , and P [8] was the most common P genotype  among the 716 rotavirus-positive specimens. G9P [8], G3P [8] and G2P [4] were the most common strains. The rotavirus-positive rates of samples in 2012 and 2013 were higher than that in 2011, and the dominant genotype changed from G3P [8] to G9P [8] in 2012 and 2013. VP7 gene sequences of G9 strains in this study clustered into two main lineages. Most of the G9 strains exhibited the highest nucleotide similarity (99.1%~ 100.0%) to the strain found in Japan (MI1128). VP4 gene sequences of P [8] strains were almost P[8]b.Rotavirus accounted for more than one fifth of childhood diarrhea in Beijing during the study period. Targeted measures such as immunization with effective rotavirus vaccines should be carried out to reduce the morbidity and mortality due to rotavirus. Infection with group A rotavirus is the main cause of severe diarrheal disease in children worldwide , and oneAccording to the sequence diversity of VP7 and VP4 genes, group A rotavirus can be divided into several G and P genotypes . So far,There is no specific drug for the rotavirus diarrhea, but effective vaccines are available and can protect children from illness and death caused by rotavirus. In China, only the LLR  vaccine is licensed for use, which is sourced from G10P [12] of sheep. However, this vaccine has not yet been included in the national immunization program, and there are no available clinical trial data so far. Further understanding the distribution of rotavirus strains may provide evidence for the use of vaccines in China. Previous data in China, usually based on a short-term observational studies, are limited. However, in this study we presented the results of 6-years of surveillance data to further elucidate the changing epidemiology, and thus inform targeted control measures and vaccine development.Data were collected on children with acute diarrhea identified through hospital-based surveillance from 2011 to 2016 in Beijing. Acute diarrhea cases were defined as outpatient children aged under 5 years old who had diarrhea (3 or more loose stools within 24 h). Children who were not residents in Beijing were excluded. Sentinel hospital surveillance for rotavirus diarrhea among children under 5 years old was established in three children’s hospitals located in Xicheng, Chaoyang and Tongzhou districts respectively. Stool specimens and socio-demographic data were collected from children under 5 years old who were newly diagnosed with acute diarrhea at the first medical consultation. Fifteen specimens were collected every month from each sentinel hospital. Socio-demographic information includes district, surveillance month, age and sex. District centers for disease control (CDC) are responsible for sending specimens and information of patients to Beijing CDC each month.Rotavirus antigen detection was performed by enzyme linked immunosorbent assay (ELISA) using ProSpecT Rotavirus Microplate Assay , according to the manufacturer’s instructions.According to the manufacturer’s protocol, QIAamp Viral RNA Mini Kit  was used to extract Viral RNA from 140 μL of a 10% fecal suspension in phosphate-buffered saline. Extracted RNA was stored at − 20 °C until further use.Rotavirus G and P genotyping were determined by multiplex semi-nested reverse transcription polymerase chain reaction (RT-PCR) , 28. A tThe second-round PCR was performed using AmpliTaq Gold® 360 PCR Master Mix . For G genotyping, primers aBT1, aCT2, G3, aDT4, aAT8, G9, G12 and VP7R were used, generating amplicons of 618 bp, 521 bp, 682 bp, 452 bp, 754 bp, 179 bp and 387 bp for G1, G2, G3, G4, G8, G9 and G12, respectively. For P genotyping, primers VP4F, 1 T-1D, 2 T-1, 3 T-1, 4 T-1, 5 T-1, and P [11] were used, generating amplicons of 362 bp, 146 bp, 224 bp, 270 bp, 462 bp and 191 bp for P [4], P [6], P [8], P [9], P [10] and P [11], respectively. PCR was performed at 95 °C for 10 min followed by 35 cycles of 95 °C for 30 s, 42 °C for 30 s, and 72 °C for 60 s; a final extension was run at 72 °C for 7 min. The PCR products were analyzed using a QIAxcel Advanced Instrument with a QIAxcel DNA Screening Kit .The first-round PCR products were purified and sequenced directly on an ABI 3730xl DNA Analyzer using a BigDye Terminator v3.1 Cycle Sequencing Kit . All sequences were prepared and aligned by BioEdit (version 7.0.9.0) with the Clustal W program. The phylogenetic tree was constructed using the neighbor-joining method with MEGA software (version 6.06) and bootstrap analysis was performed with 1000 replications. The nucleotide sequences used in this study were submitted in GenBank under accession numbers MH631063-MH631154, MH625726-MH625776 and MH625777-MH625915. The reference sequences used in plotting the phylogeny tree from the GenBank database are shown in Table P < 0.05 was considered statistically significant.Socio-demographical data and laboratory data were double entered and checked. Data analyses were performed using SPSS (version 20.0). Univariate logistic regression analyses were conducted to identify factors predictive of rotavirus infection. Multivariate logistic regression was conducted to adjust for confounders. Chi-square test was used to compare the distribution of GP genotypes between different genders, age groups, living areas and years. All hypothesis tests were 2-tailed, and a probability of A total of 3668 stool specimens of children with acute diarrhea were collected from 3668 cases, including 2296 specimens from boys, 1363 specimens from girls, 9 cases of unknown gender. A total of 762 stool specimens tested positive for rotavirus with the positive rate of 20.8%.OR 0.57, 95% CI 0.47–0.68). From 2011 to 2016, the rotavirus-positive rates were 16.4% (99/604), 27.3% (180/659), 24.6% (161/654), 17.8% (99/555), 17.1% (91/531), and 19.8% (132/665), respectively each year. The rotavirus-positive rates in 2012 and 2013 were higher, being > 20%. Controlled for age and urban residence, rotavirus-positive rates were significantly different between different years (P < 0.001). The rotavirus-positive rates in 2012 and 2013 were higher, compared with 2011. Adjusted OR was 1.90 (95% CI 1.43–2.54) in 2012 and 1.60 (95% CI 1.19–2.15) in 2013. The largest number and highest rotavirus-positive rate of specimens were in November, December and the following January each epidemic season . Children aged 1–2 years old had the highest rotavirus-positive rate (29.0%), followed by those aged 6–11 months old (24.0%), and children aged 2–3 years old (17.9%)  were genotyped successfully for G and P Table . G9 was χ2 = 4.492, P = 0.344), age group  or living area . In 2011, G3P [8] and G9P [8] were both the dominant genotypes, accounting for 24.2 (24/99) and 22.2% (22/99) of samples respectively. In 2012, G9P [8] became the dominant genotype, accounting for 56.1% (101/180) of samples. From 2013 to 2016, G9P [8] was the dominant genotype, accounting for 70%~ 80% of samples .The most common GP combined genotype was G9P [8] , followed by G3P [8] and G2P [4]. G3P [8] and G9P [8] mixed accounted for 48.4% (46/95) of the others. There was no statistically significant difference in distribution of genotype by gender  Fig. . Most ofP genotypes were identified by phylogenetic analysis of VP4 genes. In this study, the P [8] strains isolated were clustered into two minor lineages (P[8]b and P[8]c) Fig. . Most ofThe P [4] strains isolated were clustered into two minor lineages (I and II), similar to the results of Than VT et al. . PhylogeUnderstanding the distribution and antigenic variation of rotavirus infection among children under 5 years old in China can inform vaccination policy in the future. Our study showed that rotavirus was responsible for 20.8% of diarrhea in outpatient children in Beijing, ranging from 16.4 to 27.3%. Children aged 1–2 years old had the highest rotavirus-positive rate (29.0%), followed by those aged 6–11 months old (24.0%), and children aged 2–3 years old (17.9%). The circulation of rotavirus was seasonal, peaking from November to January each year in Beijing. Urban children had higher rotavirus-positive rates compared with children who lived in rural areas, probably reflecting crowding and sanitation. The data also shows significantly different rotavirus-positive rates in different years. G9P [8] had become the dominant genotype in recent years. Some lineages of VP7 and VP4 gene sequences were discovered in previous years, but not in recent years.Previous studies showed that, the rotavirus-positive rate varied among different regions and years in China. These studies showed that the rotavirus-positive rates of outpatient service ranged from 14.1 to 40.8%, with the rotavirus-positive of hospitalization was even higher in previous studies –33. Our Most children infected with rotavirus at least once before the age of 5 years old , 34. A pThere was a strong association between rotavirus infection in childhood and climate factors, with highest rotavirus-positive rates during the cold season . For eveOur research found that children who lived in rural areas had lower rotavirus-positive rates compared with urban children. This result was similar to a study in Northern Cameroon . Less crEpidemic strains vary over time and place in both developed and developing countries. G12P [8] was the dominant genotype in children in the United States during 2011 to 2013 . G1P [8]Evolutionary trees in this study were used to verify that the G and P strains were divided into different genotypes correctly. There were at least ten branches of VP7 gene sequences of G9 type, originating from people or pigs. Our study showed that G9 strains clustered into two lineages. The main lineage of G9 strains were similar to that in Japan, the United States, China and other countries. In another lineage, there were only four strains. These were similar to strains found in USA (F45) in the past. But in recent 3 years, this strain has not been circulating. The VP4 gene sequences of P [8] clustered into most P[8]b and P[8]c lineages. Only fifteen strains were P[8]c mostly before 2014, similar to the strain found in Russia (Nov10-N709). However, P[8]b was widely prevalent in Beijing in recent years. The P[8]b in Beijing was similar to that in Japan , the United States (CNMC3), China  and other countries of the world in recent years. According to previous studies , 49, somIn conclusion, our study highlights that rotavirus causes a high disease burden in children under 5 years in Beijing, with a change in predominant strains over time. At present, G9P [8] is the dominant strain. Further research is needed to explore the related factors influencing genotypes selection, and correlation between genotypes with clinical illness severity in children. The economic burden of rotavirus-positive and the cost-effectiveness of a vaccination program are also should be considered. Currently there is no specific drug for the rotavirus diarrhea, but vaccines are available and can protect children from illness and death caused by rotavirus. In 2009, WHO recommended that all countries should promote the rotavirus vaccine . RotaTeqRotavirus was a major cause of childhood diarrhea in Beijing during 2011 to 2016. The rotavirus-positive rate of 2012 and 2013 were higher than 2011, the dominant genotype changed from G3P [8] to G9P [8] in that 2 years. At present, G9P [8] is the dominant strain. Targeted measures, such as available rotavirus vaccination could be considered in China to prevent rotavirus infection and reduce the morbidity and mortality.