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.
Mass: 21903.047 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors Type: COMPLEX 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 Entity ID: all / Source: RECOMBINANT
Molecular weight
Value: 0.155 MDa / Experimental value: YES
Source (natural)
Organism: Severe acute respiratory syndrome coronavirus 2
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 78.6 K / Temperature (min): 78.5 K / Residual tilt: 10 mradians
Image recording
Average exposure time: 5 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7994 Details: Images were collected in movie-mode at 8 frames per second.
Details: The CTF correction was done by patch CTF correction. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 2334248 Details: The particles were automatically selected using blob pickier (diameter range from 100 angstroms to 150 angstroms)
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110176 / Algorithm: FOURIER SPACE 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. Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
B value: 65 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient Details: 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.
In the structure databanks used in Yorodumi, some data are registered as the other names, "COVID-19 virus" and "2019-nCoV". Here are the details of the virus and the list of structure data.
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