+Open data
-Basic information
Entry | Database: PDB / ID: 6yyt | |||||||||||||||||||||
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Title | Structure of replicating SARS-CoV-2 polymerase | |||||||||||||||||||||
Components |
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Keywords | VIRAL PROTEIN / RNA polymerase / replication / transcription / SARS-CoV-2 | |||||||||||||||||||||
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 / host cell endosome / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / 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 / SARS-CoV-2 modulates host translation machinery / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / mRNA (nucleoside-2'-O-)-methyltransferase activity / DNA helicase / 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 endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / 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 / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / 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 synthetic construct (others) | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | |||||||||||||||||||||
Authors | Hillen, H.S. / Kokic, G. / Farnung, L. / Dienemann, C. / Tegunov, D. / Cramer, P. | |||||||||||||||||||||
Funding support | Germany, 6items
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Citation | Journal: Nature / Year: 2020 Title: Structure of replicating SARS-CoV-2 polymerase. Authors: Hauke S Hillen / Goran Kokic / Lucas Farnung / Christian Dienemann / Dimitry Tegunov / Patrick Cramer / Abstract: The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. ...The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19). | |||||||||||||||||||||
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 | 6yyt.cif.gz | 270 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6yyt.ent.gz | 207.6 KB | Display | PDB format |
PDBx/mmJSON format | 6yyt.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6yyt_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 6yyt_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 6yyt_validation.xml.gz | 42.9 KB | Display | |
Data in CIF | 6yyt_validation.cif.gz | 64.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/yy/6yyt ftp://data.pdbj.org/pub/pdb/validation_reports/yy/6yyt | HTTPS FTP |
-Related structure data
Related structure data | 11007MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10409 (Title: Structure of replicating SARS-CoV-2 polymerase / Data size: 2.5 TB Data #1: Unaligned TIF movies of SARS-CoV2 RdRp in complex with nsp7, nsp8 and RNA (part 1) [micrographs - multiframe] Data #2: Unaligned TIF movies of SARS-CoV2 RdRp in complex with nsp7, nsp8 and RNA (part 2) [micrographs - multiframe] Data #3: Warp picked and extracted particles [picked particles - single frame - processed] Data #4: Reextraced particles at 1.3 A/px (for Map2 and Map3) [picked particles - single frame - processed] Data #5: Refined particles (Map 1) [picked particles - single frame - processed] Data #6: Refined particles (Map 2) [picked particles - single frame - processed] Data #7: Refined particles (Map 3) [picked particles - single frame - processed]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 107053.227 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: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases | ||||||||
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#2: Protein | Mass: 22175.309 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) / Variant (production host): RIL References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases #3: Protein | | Mass: 9521.062 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) / Variant (production host): RIL References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA ...References: UniProt: P0DTD1, ubiquitinyl hydrolase 1, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, SARS coronavirus main proteinase, RNA-directed RNA polymerase, DNA helicase, RNA helicase, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on ester bonds, Transferases; Transferring one-carbon groups; Methyltransferases #4: RNA chain | Mass: 5687.372 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #5: Chemical | Has ligand of interest | N | |
-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 |
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Molecular weight | Value: 0.186 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.4 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Details: 15 mA, Pelco EasyGlow / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.2 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8168 Details: Images were collected at 30 degree stage tilt. Non-superres. |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1300000 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 418000 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6M71 Pdb chain-ID: A / Accession code: 6M71 / Pdb chain residue range: 1-930 / Source name: PDB / Type: experimental model |