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- EMDB-15925: Cryo-electron tomogram acquired on cryo-lamella of VeroE6 cells t... -

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Basic information

Entry
Database: EMDB / ID: EMD-15925
TitleCryo-electron tomogram acquired on cryo-lamella of VeroE6 cells transfected with SARS-CoV-2 HA-nsp3-4-V5, plunge frozen at 16 hpt
Map data
Sample
  • Virus: Severe acute respiratory syndrome coronavirus 2
Keywordscomplex / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodelectron tomography / cryo EM
AuthorsZimmermann L / Chlanda P
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 5, 2022-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15925.png

Downloads & links

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Map

FileDownload / File: emd_15925.map.gz / Format: CCP4 / Size: 3.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.47 Å/pix.
x 320 pix.
= 2069.76 Å		6.47 Å/pix.
x 1920 pix.
= 12418.56 Å		6.47 Å/pix.
x 1364 pix.
= 8822.352 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.468 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1.408174 - 0.5796269
Average (Standard dev.)0.013281063 (±0.05975292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions19201364320
Spacing13641920320
CellA: 8822.352 Å / B: 12418.56 Å / C: 2069.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Severe acute respiratory syndrome coronavirus 2

EntireName: Severe acute respiratory syndrome coronavirus 2
Components
  • Virus: Severe acute respiratory syndrome coronavirus 2

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Supramolecule #1: Severe acute respiratory syndrome coronavirus 2

SupramoleculeName: Severe acute respiratory syndrome coronavirus 2 / type: virus / ID: 1 / Parent: 0 / Details: nsp3-4 gene / NCBI-ID: 2697049
Sci species name: Severe acute respiratory syndrome coronavirus 2
Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: Yes

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE
Cryo protectantNO
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 200 / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Aquilos. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 41

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