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- EMDB-11007: Structure of replicating SARS-CoV-2 polymerase -

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Entry
Database: EMDB / ID: EMD-11007
TitleStructure of replicating SARS-CoV-2 polymerase
Map data
SampleRNA-dependent RNA polymerase complex consisting of nsp12, nsp8, nsp7 and duplex RNA.:
(nsp12) x 2 / duplex RNA / nsp8, nsp7 / nsp8 / nsp7 / nucleic-acidNucleic acid / 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 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHillen HS / Kokic G / Farnung L / Dienemann C / Tegunov D / Cramer P
Funding support Germany, 6 items
OrganizationGrant numberCountry
German Research Foundation (DFG)FOR2848 Germany
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)EXC 2067/1 39072994 Germany
European Research Council (ERC)TRANSREGULON Germany
German Research Foundation (DFG)SPP2191 Germany
Volkswagen Foundation Germany
CitationJournal: 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).
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 6, 2020-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yyt
  • Surface level: 3
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11007.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesX (Sec.)Y (Row.)Z (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: 3 / Movie #1: 3
Minimum - Maximum-15.586838 - 28.571186
Average (Standard dev.)0.016349854 (±0.9739434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
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
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-15.58728.5710.016

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Supplemental data

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Segmentation: #2

Fileemd_11007_msk_1.map
Projections & Slices
AxesZYX

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Segmentation: #1

Fileemd_11007_msk_2.map
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Additional map: Map2 nsp8b focus half map 2.

Fileemd_11007_additional_1.map
AnnotationMap2_nsp8b focus half map 2.
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Additional map: Map2 nsp8b focus half map 1.

Fileemd_11007_additional_2.map
AnnotationMap2_nsp8b focus half map 1.
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Additional map: Map2 nsp8b class half map 1.

Fileemd_11007_additional_3.map
AnnotationMap2_nsp8b _class half map 1.
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Half map: Map1 core half map 2.

Fileemd_11007_half_map_1.map
AnnotationMap1_core half map 2.
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AxesZYX

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Half map: Map1 core half map 1.

Fileemd_11007_half_map_2.map
AnnotationMap1_core half map 1.
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Sample components

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Entire RNA-dependent RNA polymerase complex consisting of nsp12, nsp8, n...

EntireName: RNA-dependent RNA polymerase complex consisting of nsp12, nsp8, nsp7 and duplex RNA.
Number of components: 9

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Component #1: protein, RNA-dependent RNA polymerase complex consisting of nsp12...

ProteinName: RNA-dependent RNA polymerase complex consisting of nsp12, nsp8, nsp7 and duplex RNA.
Recombinant expression: No
MassTheoretical: 186 kDa

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Component #2: protein, nsp12

ProteinName: nsp12 / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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Component #3: protein, duplex RNA

ProteinName: duplex RNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, nsp8, nsp7

ProteinName: nsp8, nsp7 / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, nsp12

ProteinName: nsp12 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 107.053227 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)

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Component #6: protein, nsp8

ProteinName: nsp8 / Number of Copies: 2 / 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, nsp7

ProteinName: 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 product

nucleic acidName: RNA product / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UUUUCAUGCU ACGCGUAG
MassTheoretical: 5.687372 kDa
SourceSpecies: synthetic construct (others)

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Component #9: 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 solutionpH: 7.4
Support film15 mA, Pelco EasyGlow
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 105000 X (nominal), 105000 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 1500.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 8168
Details: Images were collected at 30 degree stage tilt. Non-superres.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 418000
3D reconstructionSoftware: RELION / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 6M71
Chain ID: A
Output model

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