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- EMDB-0520: SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors -

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Entry
Database: EMDB / ID: EMD-0520
TitleSARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors
Map data
SampleSARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors:
NSP12 / NSP8 / NSP7 / ligand
Function / homology
Function and homology information


modulation by virus of host autophagy / positive regulation of ubiquitin-specific protease activity / suppression by virus of host translation / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic / Lys48-specific deubiquitinase activity / ec:3.4.22.69: / induction by virus of catabolism of host mRNA / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host ISG15 activity ...modulation by virus of host autophagy / positive regulation of ubiquitin-specific protease activity / suppression by virus of host translation / mRNA methylation / RNA phosphodiester bond hydrolysis, exonucleolytic / Lys48-specific deubiquitinase activity / ec:3.4.22.69: / induction by virus of catabolism of host mRNA / host cell endoplasmic reticulum-Golgi intermediate compartment / suppression by virus of host ISG15 activity / suppression by virus of host NF-kappaB transcription factor activity / cytoplasmic viral factory / 3'-5'-exoribonuclease activity / modulation by virus of host protein ubiquitination / ec:3.1.13.-: / omega peptidase activity / suppression by virus of host IRF3 activity / transcription, RNA-templated / 7-methylguanosine mRNA capping / viral transcription / viral genome replication / positive stranded viral RNA replication / ec:2.1.1.-: / thiol-dependent ubiquitinyl hydrolase activity / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA (guanine-N7-)-methyltransferase activity / ec:3.6.4.12: / ec:3.4.19.12: / single-stranded RNA binding / methyltransferase activity / ec:3.4.22.-: / helicase activity / host cell perinuclear region of cytoplasm / viral protein processing / host cell membrane / suppression by virus of host type I interferon-mediated signaling pathway / double-stranded RNA binding / ec:3.6.4.13: / ec:2.7.7.48: / induction by virus of host autophagy / viral RNA genome replication / cysteine-type endopeptidase activity / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / ec:3.1.-.-: / transcription, DNA-templated / RNA binding / zinc ion binding / integral component of membrane / ATP binding / identical protein binding
Peptidase S1, PA clan / Non structural protein 7 / Coronavirus NSP4, C-terminal domain superfamily / Replicase polyprotein 1a/1ab / NSP1 domain superfamily / Macro domain / RNA-directed RNA polymerase, catalytic domain / Peptidase C30, Coronavirus endopeptidase / NSP8 replicase superfamily / Coronavirus non-structural protein NSP16 ...Peptidase S1, PA clan / Non structural protein 7 / Coronavirus NSP4, C-terminal domain superfamily / Replicase polyprotein 1a/1ab / NSP1 domain superfamily / Macro domain / RNA-directed RNA polymerase, catalytic domain / Peptidase C30, Coronavirus endopeptidase / NSP8 replicase superfamily / Coronavirus non-structural protein NSP16 / NSP11 / RNA polymerase, N-terminal, coronaviral / Peptidase C30/C16 / NSP9 replicase / Papain-like viral protease / NSP8 replicase / Coronavirus, polyprotein cleavage domain PL2pro superfamily / RNA synthesis protein NSP10, coronavirus / Non structural protein NSP1 / SARS coronavirus, polyprotein cleavage domain PL2pro / SARS coronavirus, non-structural protein 3, N-terminal / Non-structural protein 3, coronavirus single-stranded poly(A) binding domain / (+) RNA virus helicase core domain / Coronaviridae zinc-binding domain / P-loop containing nucleoside triphosphate hydrolase / S-adenosyl-L-methionine-dependent methyltransferase / Coronavirus nonstructural protein 4, C-terminal / Replicase polyprotein, nucleic acid-binding domain (NAR) / Coronavirus RNA synthesis protein NSP10 superfamily / Replicase NSP9 superfamily / Endoribonuclease EndoU-like / NSP7 superfamily / Nsp3, coronavirus single-stranded poly(A) binding domain superfamily / Protein of unknown function (DUF3655) / nsp7 replicase / nsp8 replicase / RNA synthesis protein NSP10 / Non structural protein Nsp1 / Single-stranded poly(A) binding domain / Coronavirus polyprotein cleavage domain / Nucleic acid-binding domain (NAR) / Coronavirus RPol N-terminus / Coronavirus nonstructural protein 4 C-terminus / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase family C16 domain profile. / Macro domain profile. / Coronavirus main protease (M-pro) domain profile. / Coronaviridae zinc-binding (CV ZBD) domain profile. / (+)RNA virus helicase core domain profile. / Papain like viral protease / nsp9 replicase / NSP11 / Coronavirus NSP16 / Macro domain / Coronavirus endopeptidase C30
Replicase polyprotein 1a / Replicase polyprotein 1ab
Biological speciesSARS coronavirus / Human SARS coronavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKirchdoerfer RN / Ward AB
CitationJournal: Nat Commun / Year: 2019
Title: Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
Authors: Robert N Kirchdoerfer / Andrew B Ward /
Abstract: Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit ...Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 Å resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy. nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.
Validation ReportPDB-ID: 6nur

SummaryFull reportAbout validation report
DateDeposition: Feb 1, 2019 / Header (metadata) release: Feb 27, 2019 / Map release: May 29, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nur
  • Surface level: 0.035
  • 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_0520.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 256 pix.
= 294.4 Å
1.15 Å/pix.
x 256 pix.
= 294.4 Å
1.15 Å/pix.
x 256 pix.
= 294.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.1621221 - 0.2418724
Average (Standard dev.)0.0000073721485 (±0.007598761)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1620.2420.000

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Supplemental data

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Mask #1

Fileemd_0520_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors

EntireName: SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors
Number of components: 5

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Component #1: protein, SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors

ProteinName: SARS-Coronavirus NSP12 bound to NSP7 and NSP8 co-factors
Recombinant expression: No
MassTheoretical: 160 kDa
SourceSpecies: SARS coronavirus
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Vector: pFastBac / Cell of expression system: Sf21

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Component #2: protein, NSP12

ProteinName: NSP12 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 109.277031 kDa
SourceSpecies: Human SARS coronavirus
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, NSP8

ProteinName: NSP8 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 21.88799 kDa
SourceSpecies: Human SARS coronavirus
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #4: protein, NSP7

ProteinName: NSP7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.333869 kDa
SourceSpecies: Human SARS coronavirus
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #5: 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.: 3.1 mg/mL
Buffer solution: n-dodecyl-beta-D-maltopyranoside was added just prior to spotting samples onto holey EM grids.
pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 43478.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1677

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 71046
3D reconstructionSoftware: RELION / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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