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- EMDB-15965: Subtomogram average of SARS-CoV-2 nsp3-4 delate Ubl1-Ubl2 obtaine... -

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Basic information

Entry
Database: EMDB / ID: EMD-15965
TitleSubtomogram average of SARS-CoV-2 nsp3-4 delate Ubl1-Ubl2 obtained from cryo-ET of cryo-FIB milled VeroE6 cells transfected with nsp3-4 delta Ubl1-Ubl2
Map dataSubtomogram average of SARS-CoV-2 nsp3-4 deltaUbl1-Ubl1 (filtered to 40A)
Sample
  • Cell: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5
Keywordsnsp3-4 protein / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsubtomogram averaging / cryo EM / Resolution: 30.0 Å
AuthorsChlanda P / Zimmermann L
Funding support Germany, 1 items
OrganizationGrant numberCountry
Other privateChica and Heinz Schaller Foundation Germany
CitationJournal: Nat Commun / Year: 2023
Title: SARS-CoV-2 nsp3 and nsp4 are minimal constituents of a pore spanning replication organelle.
Authors: Liv Zimmermann / Xiaohan Zhao / Jana Makroczyova / Moritz Wachsmuth-Melm / Vibhu Prasad / Zach Hensel / Ralf Bartenschlager / Petr Chlanda /
Abstract: Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal ...Coronavirus replication is associated with the remodeling of cellular membranes, resulting in the formation of double-membrane vesicles (DMVs). A DMV-spanning pore was identified as a putative portal for viral RNA. However, the exact components and the structure of the SARS-CoV-2 DMV pore remain to be determined. Here, we investigate the structure of the DMV pore by in situ cryo-electron tomography combined with subtomogram averaging. We identify non-structural protein (nsp) 3 and 4 as minimal components required for the formation of a DMV-spanning pore, which is dependent on nsp3-4 proteolytic cleavage. In addition, we show that Mac2-Mac3-DPUP-Ubl2 domains are critical for nsp3 oligomerization and crown integrity which influences membrane curvature required for biogenesis of DMVs. Altogether, SARS-CoV-2 nsp3-4 have a dual role by driving the biogenesis of replication organelles and assembly of DMV-spanning pores which we propose here to term replicopores.
History
DepositionOct 15, 2022-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15965.png

Downloads & links

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Map

FileDownload / File: emd_15965.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of SARS-CoV-2 nsp3-4 deltaUbl1-Ubl1 (filtered to 40A)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 256 pix.
= 551.936 Å		2.16 Å/pix.
x 256 pix.
= 551.936 Å		2.16 Å/pix.
x 256 pix.
= 551.936 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.325
Minimum - Maximum-2.16653 - 2.6359339
Average (Standard dev.)0.024209874 (±0.50565505)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 551.936 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15965_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15965_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA...

EntireName: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5
Components
  • Cell: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5

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Supramolecule #1: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA...

SupramoleculeName: VeroE6 cells transfected with SARS-CoV-2 nsp3-4 truncation: pCDNA3.1-HA-nsp3-deltaUbl1-Ubl2-nsp4-V5
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 42000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo (ver. 1.1.514) / Number subtomograms used: 94
ExtractionNumber tomograms: 8 / Number images used: 94 / Software - Name: Dynamo (ver. 1.1.514)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Dynamo (ver. 1.1.514)
FSC plot (resolution estimation)

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