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Yorodumi- PDB-7bzf: COVID-19 RNA-dependent RNA polymerase post-translocated catalytic... -
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-Basic information
Entry | Database: PDB / ID: 7bzf | ||||||||||||||||||||||||
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Title | COVID-19 RNA-dependent RNA polymerase post-translocated catalytic complex | ||||||||||||||||||||||||
Components |
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Keywords | VIRAL PROTEIN/RNA / COVID-19 / 2019-nCoV / SARS-CoV-2 / Virus / RdRp / nsp12 / nsp7 / nsp8 / RTC / cryo-EM / Viral protein / RNA polymerase / drug target / antiviral / replication transcription complex / VIRAL PROTEIN-RNA complex | ||||||||||||||||||||||||
Function / homology | Function and homology information protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 5'-3' DNA helicase activity / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / induction by virus of host autophagy / copper ion binding / cysteine-type endopeptidase activity / RNA-directed RNA polymerase / viral RNA genome replication / virus-mediated perturbation of host defense response / RNA-dependent RNA polymerase activity / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane Similarity search - Function | ||||||||||||||||||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 Foot-and-mouth disease virus | ||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å | ||||||||||||||||||||||||
Authors | Wang, Q. / Gao, Y. / Ji, W. / Mu, A. / Rao, Z. | ||||||||||||||||||||||||
Funding support | China, 7items
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Citation | Journal: Cell / Year: 2020 Title: Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase. Authors: Quan Wang / Jiqin Wu / Haofeng Wang / Yan Gao / Qiaojie Liu / An Mu / Wenxin Ji / Liming Yan / Yan Zhu / Chen Zhu / Xiang Fang / Xiaobao Yang / Yucen Huang / Hailong Gao / Fengjiang Liu / Ji ...Authors: Quan Wang / Jiqin Wu / Haofeng Wang / Yan Gao / Qiaojie Liu / An Mu / Wenxin Ji / Liming Yan / Yan Zhu / Chen Zhu / Xiang Fang / Xiaobao Yang / Yucen Huang / Hailong Gao / Fengjiang Liu / Ji Ge / Qianqian Sun / Xiuna Yang / Wenqing Xu / Zhijie Liu / Haitao Yang / Zhiyong Lou / Biao Jiang / Luke W Guddat / Peng Gong / Zihe Rao / Abstract: Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete ...Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery. | ||||||||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7bzf.cif.gz | 237.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7bzf.ent.gz | 182.2 KB | Display | PDB format |
PDBx/mmJSON format | 7bzf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7bzf_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7bzf_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 7bzf_validation.xml.gz | 45.2 KB | Display | |
Data in CIF | 7bzf_validation.cif.gz | 66.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bz/7bzf ftp://data.pdbj.org/pub/pdb/validation_reports/bz/7bzf | HTTPS FTP |
-Related structure data
Related structure data | 30252MC 7c2kC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Non-structural protein ... , 2 types, 3 molecules BDC
#2: Protein | 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 Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1 #3: Protein | | Mass: 9248.804 Da / Num. of mol.: 1 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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-RNA chain , 2 types, 2 molecules FG
#4: RNA chain | Mass: 10038.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Foot-and-mouth disease virus |
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#5: RNA chain | Mass: 4446.708 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Foot-and-mouth disease virus |
-Protein / Non-polymers , 2 types, 3 molecules A
#1: Protein | Mass: 108162.461 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0DTD1, RNA-directed RNA polymerase |
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#6: Chemical |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Structure of COVID-19 RNA-dependent RNA polymerase in complex with RNA Type: COMPLEX Details: full-length COVID-19 nsp12 (residues S1-Q932) was incubated with nsp7 (residues S1-Q83) and nsp8 (A1-Q198), and in complex with RNA template and product. Entity ID: #1-#5 / Source: RECOMBINANT |
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Molecular weight | Value: 0.155 MDa / Experimental value: YES |
Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono disperse. |
Specimen support | Grid type: Quantifoil R0.6/1 |
Vitrification | Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Preliminary grid screening was preformed manually. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 78.5 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: 8494 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Chromatic aberration corrector: None / Energyfilter slit width: 40 eV / Spherical aberration corrector: None. |
Image scans | Sampling size: 2.5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 2-40 |
-Processing
Software | Name: PHENIX / Version: 1.16_3549: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: The selected images were normalized. | ||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTF correction was done by patch CTF correction. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1235162 | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119662 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 58.5 / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient | ||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Accession code: 6NUR / Initial refinement model-ID: 1 / PDB-ID: 6NUR / Source name: PDB / Type: experimental model
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Refine LS restraints |
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