National Natural Science Foundation of China (NSFC)
81520108019
China
National Natural Science Foundation of China (NSFC)
813300237
China
Ministry of Science and Technology (MoST, China)
2017YFC0840300
China
Citation
Journal: Science / Year: 2020 Title: Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Authors: Yan Gao / Liming Yan / Yucen Huang / Fengjiang Liu / Yao Zhao / Lin Cao / Tao Wang / Qianqian Sun / Zhenhua Ming / Lianqi Zhang / Ji Ge / Litao Zheng / Ying Zhang / Haofeng Wang / Yan Zhu / ...Authors: Yan Gao / Liming Yan / Yucen Huang / Fengjiang Liu / Yao Zhao / Lin Cao / Tao Wang / Qianqian Sun / Zhenhua Ming / Lianqi Zhang / Ji Ge / Litao Zheng / Ying Zhang / Haofeng Wang / Yan Zhu / Chen Zhu / Tianyu Hu / Tian Hua / Bing Zhang / Xiuna Yang / Jun Li / Haitao Yang / Zhijie Liu / Wenqing Xu / Luke W Guddat / Quan Wang / Zhiyong Lou / Zihe Rao / Abstract: A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of ...A novel coronavirus [severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)] outbreak has caused a global coronavirus disease 2019 (COVID-19) pandemic, resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase [(RdRp), also named nsp12] is the central component of coronaviral replication and transcription machinery, and it appears to be a primary target for the antiviral drug remdesivir. We report the cryo-electron microscopy structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-angstrom resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics that target viral RdRp.
History
Deposition
Mar 16, 2020
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Header (metadata) release
Mar 25, 2020
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Map release
Apr 1, 2020
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Update
Mar 10, 2021
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Current status
Mar 10, 2021
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors
Entire
Name: SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors
Components
Complex: SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors
Protein or peptide: RNA-directed RNA polymerase
Protein or peptide: Non-structural protein 7
Protein or peptide: Non-structural protein 8
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Supramolecule #1: SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors
Supramolecule
Name: SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: The bacterially expressed full-length SARS-CoV-2 nsp12 (residues S1-Q932) was incubated with nsp7 (residues S1-Q83) and nsp8 (residues A1-Q198), and the complex was then purified
Source (natural)
Organism: Severe acute respiratory syndrome coronavirus 2
Details: Solutions were made fresh from concentrated to avoid microbial contamination.
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa / Details: None.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK II / Details: blot for 3 seconds before plunging..
Details
This sample was mono disperse.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Min: 78.5 K / Max: 78.6 K
Alignment procedure
Coma free - Residual tilt: 10.0 mrad
Specialist optics
Phase plate: VOLTA PHASE PLATE / Chromatic aberration corrector: NONE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
Details
Preliminary grid screening was performed manually.
Image recording
Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 2.5 µm / Digitization - Frames/image: 2-40 / Number grids imaged: 2 / Number real images: 7994 / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2 Details: Images were collected in movie-mode at 8 frames per second.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Number selected: 2334248 Details: The particles were automatically selected using blob pickier (diameter range from 100 angstroms to 150 angstroms)
CTF correction
Software - Name: cryoSPARC (ver. 2.9.1) Software - details: cryoSPARC was used to determine CTF correction. Details: The CTF correction was done by patch CTF correction.
Startup model
Type of model: NONE / Details: The startup models were generated automatically.
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9.1) Software - details: cryoSPARC was used to do final reconstruction Details: The particle set was used to perform homogeneous refinement, yielding a resolution of 3.1-angstrom. After local refinement, the final resolution reached 2.9-angstrom. Number images used: 110176
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.1) Software - details: cryoSPARC was used to do initial angular assignment Details: The initial angle assignment was generated by Ab-initio reconstruction.
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.1) Software - details: cryoSPARC was used to do final angular assignment. Details: The final angle assignment was non-uniform refinement.
Final 3D classification
Number classes: 5 / Avg.num./class: 183622 / Software - Name: cryoSPARC (ver. 2.9.1) Software - details: cryoSPARC was used to do final classification Details: The final 3D classification can distinguish one class matching complete complex.
After the corresponding amino acids were replaced with those from 2019-nCoV, the model was manually built in Coot with the guidance of the cryo-EM map, and in combination with real space refinement with Phenix.
Refinement
Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 65 / Target criteria: correlation coefficient
Output model
PDB-6m71: SARS-Cov-2 RNA-dependent RNA polymerase in complex with cofactors
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