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- EMDB-11993: Structure of elongating SARS-CoV-2 RNA-dependent RNA polymerase w... -

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Basic information

Entry
Database: EMDB / ID: EMD-11993
TitleStructure of elongating SARS-CoV-2 RNA-dependent RNA polymerase with Remdesivir at position -3 (structure 1)
Map data
SampleSARS-CoV-2 RNA-dependent RNA polymerase complex (nsp12, nsp7, nsp8) with elongation scaffold containing remdesivir at position -3.
  • (SARS-CoV-2 RNA-dependent RNA polymerase ...) x 2
  • SARS-CoV-2 nsp7 and nsp8
  • DNA and RNA
  • SARS-CoV-2 nsp8
  • SARS-CoV-2 nsp7
  • (nucleic-acidNucleic acid) x 2
  • ligand
Function / homology
Function and homology information


Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Translation of Replicase and Assembly of the Replication Transcription Complex / 5'-3' RNA helicase activity / RNA phosphodiester bond hydrolysis, exonucleolytic / Lyases; Phosphorus-oxygen lyases / modulation by virus of host autophagy ...Maturation of replicase proteins / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Transcription of SARS-CoV-2 sgRNAs / host cell endosome / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Translation of Replicase and Assembly of the Replication Transcription Complex / 5'-3' RNA helicase activity / RNA phosphodiester bond hydrolysis, exonucleolytic / Lyases; Phosphorus-oxygen lyases / modulation by virus of host autophagy / mRNA methylation / double membrane vesicle viral factory outer membrane / suppression by virus of host translation / ISG15-specific protease activity / host cell Golgi apparatus / Replication of the SARS-CoV-2 genome / suppression by virus of host type I interferon production / host cell endoplasmic reticulum / SARS coronavirus main proteinase / induction by virus of catabolism of host mRNA / cytoplasmic viral factory / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 3'-5'-exoribonuclease activity / G-quadruplex RNA binding / suppression by virus of host ISG15-protein conjugation / host cell endoplasmic reticulum-Golgi intermediate compartment / protein K48-linked deubiquitination / suppression by virus of host toll-like receptor signaling pathway / suppression by virus of host viral-induced cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / transcription, RNA-templated / suppression by virus of host NF-kappaB cascade / viral transcription / modulation by virus of host protein ubiquitination / protein K63-linked deubiquitination / methyltransferase cap1 / positive stranded viral RNA replication / protein autoprocessing / cysteine-type peptidase activity / viral genome replication / mRNA (nucleoside-2'-O-)-methyltransferase activity / suppression by virus of host TRAF activity / helicase activity / ubiquitinyl hydrolase 1 / DNA helicase / thiol-dependent deubiquitinase / DNA helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / viral protein processing / RNA helicase / induction by virus of host autophagy / endonuclease activity / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / suppression by virus of host type I interferon-mediated signaling pathway / transcription, DNA-templated / host cell cytoplasm / protein dimerization activity / : / protein homodimerization activity / zinc ion binding / integral component of membrane / ATP binding / identical protein binding / cytosol
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Nonstructural protein 14, betacoronavirus / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Viral (Superfamily 1) RNA helicase / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerisation / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Coronavirus proofreading exoribonuclease / Coronavirus 2'-O-methyltransferase / Coronavirus replicase NSP15, middle domain / Coronavirus RNA-dependent RNA polymerase, N-terminal / Coronavirus replicase NSP15, N-terminal oligomerisation / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Coronavirus replicase NSP15, middle domain / Coronaviridae zinc-binding (CV ZBD) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP1 superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, coronavirus / Non-structural protein 2, SARS-CoV-like / Betacoronavirus replicase NSP3, N-terminal / Betacoronavirus SUD-C domain / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / NendoU domain, nidovirus / Endoribonuclease EndoU-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / (+)RNA virus helicase core domain profile. / (+) RNA virus helicase core domain / Betacoronavirus Nsp3c-M domain profile. / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP1, betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / Betacoronavirus Nsp3c-C domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein 6, betacoronavirus / Replicase polyprotein, nucleic acid-binding domain superfamily / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3A domain-like superfamily / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Papain-like protease, N-terminal domain superfamily, coronavirus / Lipocalin signature. / Papain-like viral protease, palm and finger domains, coronavirus / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, N-terminal / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Papain-like protease, thumb domain superfamily, coronavirus / Peptidase family C16 domain profile. / Coronavirus replicase NSP7 / Non-structural protein NSP7, coronavirus / Coronavirus endopeptidase C30 / Coronavirus papain-like peptidase / Coronavirus replicase NSP8 / Coronavirus RNA synthesis protein NSP10 / RNA synthesis protein NSP10, coronavirus / Non-structural protein 6, coronavirus / Coronavirus replicase NSP4, N-terminal / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP9, coronavirus / Coronavirus replicase NSP3, C-terminal / Coronavirus replicase NSP4, N-terminal / Non-structural protein NSP4, C-terminal, coronavirus / Coronavirus replicase NSP4, C-terminal / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Peptidase C30, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Peptidase C16, coronavirus / Coronavirus main protease (M-pro) domain profile.
Similarity search - Domain/homology
Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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.
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: 0.0002
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7b3b
  • Surface level: 0.0002
  • 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_11993.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: 0.0002 / Movie #1: 0.0002
Minimum - Maximum-0.0011212492 - 0.002032956
Average (Standard dev.)2.7598699e-06 (±4.564599e-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_11993_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_11993_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_11993_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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 remdesivir at position -3.
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 remdesivir at position -3.
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 nsp8

ProteinName: SARS-CoV-2 nsp7 and nsp8 / 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, DNA/RNA (5'-R(P*CP*UP*AP*CP*GP*CP*G)-D(P*(RMP))-R(P...

nucleic acidName: DNA/RNA (5'-R(P*CP*UP*AP*CP*GP*CP*G)-D(P*(RMP))-R(P*UP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UGAGCCUACG CG(F86)UG
MassTheoretical: 4.831936 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2

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

nucleic acidName: RNA (5'-R(P*UP*GP*CP*AP*UP*CP*GP*CP*GP*UP*AP*G)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UUUUCAUGCA UCGCGUAGGC 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: 653972
3D reconstructionAlgorithm: BACK PROJECTION / Resolution: 3.1 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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