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Yorodumi- PDB-7b3c: Structure of elongating SARS-CoV-2 RNA-dependent RNA polymerase w... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7b3c | ||||||||||||||||||||||||
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Title | Structure of elongating SARS-CoV-2 RNA-dependent RNA polymerase with Remdesivir at position -4 (structure 2) | ||||||||||||||||||||||||
Components |
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Keywords | VIRAL PROTEIN / SARS-CoV-2 / Polymerase / Transcription / Replication / RNA / Remdesivir / Inhibition / Nucleotide Analogue | ||||||||||||||||||||||||
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 | ||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||||||||||||||||||||
Authors | Kokic, G. / Hillen, H.S. / Tegunov, D. / Dienemann, C. / Seitz, F. / Schmitzova, J. / Farnung, L. / Siewert, A. / Hoebartner, C. / Cramer, P. | ||||||||||||||||||||||||
Funding support | Germany, 7items
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Citation | Journal: Nat Commun / Year: 2021 Title: Mechanism of SARS-CoV-2 polymerase stalling by remdesivir. Authors: Goran Kokic / Hauke S Hillen / Dimitry Tegunov / Christian Dienemann / Florian Seitz / Jana Schmitzova / Lucas Farnung / Aaron Siewert / Claudia Höbartner / Patrick Cramer / Abstract: Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of ...Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication. | ||||||||||||||||||||||||
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 | 7b3c.cif.gz | 209 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7b3c.ent.gz | 156.3 KB | Display | PDB format |
PDBx/mmJSON format | 7b3c.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7b3c_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7b3c_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7b3c_validation.xml.gz | 35.4 KB | Display | |
Data in CIF | 7b3c_validation.cif.gz | 52.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/b3/7b3c ftp://data.pdbj.org/pub/pdb/validation_reports/b3/7b3c | HTTPS FTP |
-Related structure data
Related structure data | 11994MC 7b3bC 7b3dC 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, 2 molecules BC
#2: Protein | Mass: 22175.309 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 |
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#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 |
-RNA chain , 2 types, 2 molecules PT
#4: RNA chain | Mass: 4831.936 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) Severe acute respiratory syndrome coronavirus 2 |
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#5: RNA chain | Mass: 18138.658 Da / Num. of mol.: 1 / Source method: obtained synthetically Source: (synth.) Severe acute respiratory syndrome coronavirus 2 |
-Protein / Non-polymers , 2 types, 3 molecules A
#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: Trichoplusia ni (cabbage looper) / 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
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Molecular weight | 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 | Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: 30 deg tilt. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: OTHER / Nominal magnification: 105000 X / Calibrated defocus min: 0.4 nm / Calibrated defocus max: 1.7 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 | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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: 1767915 | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 474061 / Details: M was used / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6YYT Accession code: 6YYT / Source name: PDB / Type: experimental model |