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- EMDB-22271: Structure of SARS-CoV-2 replication/transcription complex bound t... -

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Entry
Database: EMDB / ID: EMD-22271
TitleStructure of SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - (nsp13(2)-RTC)(2)
Map data
Sample
  • Complex: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - (nsp13(2)-RTC)2
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 7.9 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: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22271.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.21382433 - 0.7161662
Average (Standard dev.)0.0059194705 (±0.046338033)
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-0.2140.7160.006

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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(2)-RTC)2
Components
  • Complex: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - (nsp13(2)-RTC)2

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Supramolecule #1: SARS-CoV-2 replication/transcription complex bound to nsp13 helic...

SupramoleculeName: SARS-CoV-2 replication/transcription complex bound to nsp13 helicase - (nsp13(2)-RTC)2
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 65.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11771
FSC plot (resolution estimation)

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