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- EMDB-22270: Structure of SARS-CoV-2 replication/transcription complex bound t... -

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Basic information

Entry
Database: EMDB / ID: EMD-22270
TitleStructure of SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(1)-RTC
Map data
SampleSARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(1)-RTC
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsChen J / Malone B / Llewellyn EC / Campbell EA / Darst SA
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: bioRxiv / Year: 2020
Title: Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex.
Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Ed Eng / Hasan Vatandaslar / Brian T Chait / Tarun Kapoor / ...Authors: James Chen / Brandon Malone / Eliza Llewellyn / Michael Grasso / Patrick M M Shelton / Paul Dominic B Olinares / Kashyap Maruthi / Ed Eng / Hasan Vatandaslar / Brian T Chait / Tarun Kapoor / Seth A Darst / Elizabeth A Campbell /
Abstract: SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a ...SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
History
DepositionJul 3, 2020-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.28
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.28
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_22270.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 352. Å
1.1 Å/pix.
x 320 pix.
= 352. Å
1.1 Å/pix.
x 320 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.28
Minimum - Maximum-0.37648278 - 1.1179569
Average (Standard dev.)0.0021755546 (±0.041545697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.3761.1180.002

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Supplemental data

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Sample components

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Entire SARS-CoV-2 replication/transcription complex bound to nsp13 helic...

EntireName: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(1)-RTC
Number of components: 1

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Component #1: protein, SARS-CoV-2 replication/transcription complex bound to ns...

ProteinName: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - nsp13(1)-RTC
Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationCryogen name: ETHANE

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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: 65 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 17345
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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