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- PDB-7b3c: Structure of elongating SARS-CoV-2 RNA-dependent RNA polymerase w... -

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Basic information

Entry
Database: PDB / ID: 7b3c
TitleStructure of elongating SARS-CoV-2 RNA-dependent RNA polymerase with Remdesivir at position -4 (structure 2)
Components
  • DNA/RNA (5'-R(P*CP*UP*AP*CP*GP*CP*A)-D(P*(RMP))-R(P*GP*UP*G)-3')
  • RNA (5'-R(P*UP*CP*AP*CP*UP*UP*GP*CP*GP*UP*AP*G)-3')
  • SARS-CoV-2 RNA-dependent RNA polymerase (nsp12)
  • SARS-CoV-2 nsp7
  • SARS-CoV-2 nsp8
KeywordsVIRAL PROTEIN / SARS-CoV-2 / Polymerase / Transcription / Replication / RNA / Remdesivir / Inhibition / Nucleotide Analogue
Function / homology
Function and homology information


modulation by virus of host autophagy / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic / suppression by virus of host translation / ISG15-specific protease activity / suppression by virus of host type I interferon production / suppression by virus of host toll-like receptor signaling pathway / induction by virus of catabolism of host mRNA / SARS coronavirus main proteinase / cytoplasmic viral factory ...modulation by virus of host autophagy / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic / suppression by virus of host translation / ISG15-specific protease activity / suppression by virus of host type I interferon production / suppression by virus of host toll-like receptor signaling pathway / induction by virus of catabolism of host mRNA / SARS coronavirus main proteinase / cytoplasmic viral factory / suppression by virus of host ISG15 activity / protein K48-linked deubiquitination / 3'-5'-exoribonuclease activity / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host IRF3 activity / exonuclease activity / modulation by virus of host protein ubiquitination / suppression by virus of host NF-kappaB transcription factor activity / transcription, RNA-templated / protein K63-linked deubiquitination / viral genome replication / positive stranded viral RNA replication / protein autoprocessing / viral transcription / Transferases; Transferring one-carbon groups; Methyltransferases / suppression by virus of host TRAF activity / mRNA (nucleoside-2'-O-)-methyltransferase activity / helicase activity / host cell membrane / ubiquitinyl hydrolase 1 / cysteine-type peptidase activity / DNA helicase / methyltransferase activity / DNA helicase activity / single-stranded RNA binding / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / thiol-dependent deubiquitinase / host cell perinuclear region of cytoplasm / viral protein processing / methylation / RNA helicase / induction by virus of host autophagy / suppression by virus of host type I interferon-mediated signaling pathway / RNA-directed RNA polymerase / endonuclease activity / cysteine-type endopeptidase activity / RNA-directed 5'-3' RNA polymerase activity / viral RNA genome replication / RNA helicase activity / transcription, DNA-templated / host cell cytoplasm / Hydrolases; Acting on ester bonds / protein dimerization activity / protein homodimerization activity / zinc ion binding / integral component of membrane / ATP binding / identical protein binding
Coronavirus replicase NSP7 / Non-structural protein NSP3, N-terminal, betacoronavirus / Non-structural protein NSP8 superfamily, coronavirus / Endoribonuclease EndoU-like / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP3, nucleic acid-binding (NAR) domain, betacoronavirus / Non-structural protein NSP4, C-terminal, coronavirus / S-adenosyl-L-methionine-dependent methyltransferase ...Coronavirus replicase NSP7 / Non-structural protein NSP3, N-terminal, betacoronavirus / Non-structural protein NSP8 superfamily, coronavirus / Endoribonuclease EndoU-like / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP3, nucleic acid-binding (NAR) domain, betacoronavirus / Non-structural protein NSP4, C-terminal, coronavirus / S-adenosyl-L-methionine-dependent methyltransferase / P-loop containing nucleoside triphosphate hydrolase / (+) RNA virus helicase core domain / Non-structural protein NSP3, SUD-M domain, betacoronavirus / DPUP/SUD, C-terminal, betacoronavirus / RNA-directed RNA polymerase, C-terminal domain / Non-structural protein NSP1, betacoronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP8, coronavirus-like / Non-structural protein NSP7, coronavirus / Non-structural protein NSP9, coronavirus / Peptidase C16, coronavirus / RNA polymerase, N-terminal, coronaviral / Non-structural protein 14, coronavirus / Non-structural protein NSP16, coronavirus-like / Peptidase S1, PA clan / Peptidase C30, coronavirus / Macro domain / Non-structural protein NSP1 superfamily, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / Coronavirus replicase NSP8 / Coronavirus RNA-dependent RNA polymerase, N-terminal / Viral RNA-dependent RNA polymerase / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP4, N-terminal / Coronavirus replicase NSP3, C-terminal / Non-structural protein 6, coronavirus / NendoU domain, nidovirus / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Peptidase S1, PA clan, chymotrypsin-like fold / DNA/RNA polymerase superfamily / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Peptidase C30, domain 3, coronavirus / Yro2-like, 7TM domain / Macro domain-like / Papain-like protease, thumb domain superfamily, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / DNA2/NAM7 helicase-like, C-terminal / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus
Replicase polyprotein 1ab
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKokic, G. / Hillen, H.S. / Tegunov, D. / Dienemann, C. / Seitz, F. / Schmitzova, J. / Farnung, L. / Siewert, A. / Hoebartner, C. / Cramer, P.
Funding support Germany, 7items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2848 Germany
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)SPP2191 Germany
German Research Foundation (DFG)EXC 2067/1- 390729940 Germany
German Research Foundation (DFG)SPP1784 Germany
European Research Council (ERC)682586 Germany
European Research Council (ERC)693023 Germany
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 30, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 3, 2021Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: SARS-CoV-2 RNA-dependent RNA polymerase (nsp12)
B: SARS-CoV-2 nsp8
C: SARS-CoV-2 nsp7
P: DNA/RNA (5'-R(P*CP*UP*AP*CP*GP*CP*A)-D(P*(RMP))-R(P*GP*UP*G)-3')
T: RNA (5'-R(P*UP*CP*AP*CP*UP*UP*GP*CP*GP*UP*AP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,8517
Polymers161,7205
Non-polymers1312
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11650 Å2
ΔGint-95 kcal/mol
Surface area40770 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein SARS-CoV-2 RNA-dependent RNA polymerase (nsp12) / pp1ab / ORF1ab polyprotein


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, 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
#2: Protein SARS-CoV-2 nsp8 / pp1ab / ORF1ab polyprotein


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, 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 SARS-CoV-2 nsp7 / pp1ab / ORF1ab polyprotein


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

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RNA chain , 2 types, 2 molecules PT

#4: RNA chain DNA/RNA (5'-R(P*CP*UP*AP*CP*GP*CP*A)-D(P*(RMP))-R(P*GP*UP*G)-3')


Mass: 4831.936 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2
#5: RNA chain RNA (5'-R(P*UP*CP*AP*CP*UP*UP*GP*CP*GP*UP*AP*G)-3')


Mass: 18138.658 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Severe acute respiratory syndrome coronavirus 2

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Non-polymers , 1 types, 2 molecules

#6: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1SARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp8) with elongation scaffold containing remdesivir at position -4.COMPLEX#1-#50MULTIPLE SOURCES
2SARS-CoV-2 RNA-dependent RNA polymerase (nsp12)COMPLEX#11RECOMBINANT
3SARS-CoV-2 nsp7 and 8COMPLEX#2-#31RECOMBINANT
4DNA and RNACOMPLEX#4-#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Severe acute respiratory syndrome coronavirus 22697049
23Severe acute respiratory syndrome coronavirus 22697049
34Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Escherichia coli BL21(DE3) (bacteria)469008
34synthetic construct (others)32630
Buffer solutionpH: 7.4
Buffer component
IDConc.FormulaBuffer-ID
120 mMHepes1
2100 mMNaClSodium chloride1
31 mMMgCl21
41 mMTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: 30 deg tilt.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: 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 holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Warp1.0.7particle selection
2SerialEM3.8image acquisition
4Warp1.0.7CTF correction
7Coot0.9model fitting
9PHENIX1.18model refinement
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1767915
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 474061 / Details: M was used / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6YYT

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