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- EMDB-11995: Structure of elongating SARS-CoV-2 RNA-dependent RNA polymerase w... -

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Entry
Database: EMDB / ID: EMD-11995
TitleStructure of elongating SARS-CoV-2 RNA-dependent RNA polymerase with AMP at position -4 (structure 3)
Map data
SampleSARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp8) with elongation scaffold containing AMP at position -4.
  • (SARS-CoV-2 RNA-dependent RNA polymerase ...) x 2
  • SARS-CoV-2 nsp7 and 8
  • DNA and RNA
  • SARS-CoV-2 nsp8
  • SARS-CoV-2 nsp7
  • (nucleic-acidNucleic acid) x 2
  • ligand
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
P-loop containing nucleoside triphosphate hydrolase / Peptidase C30, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Non-structural protein NSP3A domain-like superfamily / S-adenosyl-L-methionine-dependent methyltransferase / 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 ...P-loop containing nucleoside triphosphate hydrolase / Peptidase C30, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Non-structural protein NSP3A domain-like superfamily / S-adenosyl-L-methionine-dependent methyltransferase / 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 / RNA-directed RNA polymerase, C-terminal domain / Macro domain / Peptidase S1, PA clan / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronaviral / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP7, coronavirus / Non-structural protein NSP8, coronavirus-like / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP1, betacoronavirus / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / (+) RNA virus helicase core domain / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein NSP1 superfamily, betacoronavirus / Yro2-like, 7TM domain / Coronavirus replicase NSP15, middle domain / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / DNA/RNA polymerase superfamily / Papain-like viral protease, palm and finger domains, coronavirus / Non-structural protein NSP15, middle domain superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / Coronavirus replicase NSP15, N-terminal oligomerisation / NendoU domain, nidovirus / Peptidase C30, domain 3, coronavirus / Non-structural protein 6, coronavirus / Coronavirus replicase NSP3, C-terminal / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP4, N-terminal / Non-structural protein 2, C-terminal domain, coronavirus / Nonstructural protein 2, N-terminal domain, coronavirus / DNA2/NAM7 helicase-like, C-terminal / Macro domain-like
Replicase polyprotein 1ab
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKokic G / Hillen HS / Tegunov D / Dienemann C / Seitz F / Schmitzova J / Farnung L / Siewert A / Hoebartner C / Cramer P
Funding support Germany, 7 items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2848 Germany
German Research Foundation (DFG)SPP2191 Germany
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)SPP1784 Germany
German Research Foundation (DFG)EXC 2067/1- 390729940 Germany
European Research Council (ERC)693023 Germany
European Research Council (ERC)682586 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 ReportSummary, Full report, XML, About validation report
History
DepositionNov 30, 2020-
Header (metadata) releaseDec 23, 2020-
Map releaseDec 23, 2020-
UpdateFeb 3, 2021-
Current statusFeb 3, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7b3d
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11995.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 240 pix.
= 200.16 Å
0.83 Å/pix.
x 240 pix.
= 200.16 Å
0.83 Å/pix.
x 240 pix.
= 200.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.0003 / Movie #1: 0.0003
Minimum - Maximum-0.00090332614 - 0.0017360445
Average (Standard dev.)2.1078765e-06 (±4.1083505e-05)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 200.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z200.160200.160200.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0010.0020.000

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Supplemental data

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Segmentation: #1

Fileemd_11995_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Half map: Half map 2

Fileemd_11995_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_11995_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
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Sample components

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Entire SARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp...

EntireName: SARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp8) with elongation scaffold containing AMP at position -4.
Number of components: 10

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Component #1: protein, SARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, ...

ProteinName: SARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp8) with elongation scaffold containing AMP at position -4.
Recombinant expression: No

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Component #2: protein, SARS-CoV-2 RNA-dependent RNA polymerase nsp12

ProteinName: SARS-CoV-2 RNA-dependent RNA polymerase nsp12 / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #3: protein, SARS-CoV-2 nsp7 and 8

ProteinName: SARS-CoV-2 nsp7 and 8 / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, DNA and RNA

ProteinName: DNA and RNA / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: synthetic construct (others)

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Component #5: protein, SARS-CoV-2 RNA-dependent RNA polymerase nsp12

ProteinName: SARS-CoV-2 RNA-dependent RNA polymerase nsp12 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 107.053227 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #6: protein, SARS-CoV-2 nsp8

ProteinName: SARS-CoV-2 nsp8 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 22.175309 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: protein, SARS-CoV-2 nsp7

ProteinName: SARS-CoV-2 nsp7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.521062 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #8: nucleic-acid, RNA (5'-R(P*CP*UP*AP*CP*GP*CP*AP*GP*UP*G)-3')

nucleic acidName: RNA (5'-R(P*CP*UP*AP*CP*GP*CP*AP*GP*UP*G)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UGAGCCUACG CAGUG
MassTheoretical: 4.807914 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2

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Component #9: nucleic-acid, RNA (5'-R(P*UP*GP*CP*AP*CP*UP*GP*CP*GP*UP*AP*G)-3')

nucleic acidName: RNA (5'-R(P*UP*GP*CP*AP*CP*UP*GP*CP*GP*UP*AP*G)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UUUUCAUGCA CUGCGUAGGC UCAUACCGUA UUGAGACCUU UUGGUCUCAA UACGGUA
MassTheoretical: 18.138658 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2

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Component #10: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: 30 deg tilt.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: OTHER
LensMagnification: 105000 X (nominal) / Cs: 2.7 mm / Imaging mode: OTHER / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 819273
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Details: M was used

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 6YYT
Output model

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