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- EMDB-39111: SARS-CoV-2 DMV nsp3-4 pore complex (extended-pore) -

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Basic information

Entry
Database: EMDB / ID: EMD-39111
TitleSARS-CoV-2 DMV nsp3-4 pore complex (extended-pore)
Map datalocal resolution map
Sample
  • Complex: SARS-CoV-2 nsp3-4 pore complex
KeywordsDouble membrane vesicle / pore complex / nsp3 / nsp4 / RNA transport / VIRAL PROTEIN
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsubtomogram averaging / cryo EM / Resolution: 4.7 Å
AuthorsHuang YX / Zhong LJ / Zhang WX / Ni T
Funding support Hong Kong, 1 items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC) Hong Kong
CitationJournal: Nature / Year: 2024
Title: Molecular architecture of coronavirus double-membrane vesicle pore complex.
Authors: Yixin Huang / Tongyun Wang / Lijie Zhong / Wenxin Zhang / Yu Zhang / Xiulian Yu / Shuofeng Yuan / Tao Ni /
Abstract: Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications. SARS-CoV-2 non-structural ...Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications. SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs. The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3-nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3-nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection.
History
DepositionFeb 12, 2024-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateSep 18, 2024-
Current statusSep 18, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39111.png

Downloads & links

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Map

FileDownload / File: emd_39111.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal resolution map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.5711.5711.571
CCP4 map header1.881.881.88
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-4.083556 - 6.000817
Average (Standard dev.)0.0000015573632 (±0.12533948)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 452.448 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39111_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_39111_half_map_1.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 : SARS-CoV-2 nsp3-4 pore complex

EntireName: SARS-CoV-2 nsp3-4 pore complex
Components
  • Complex: SARS-CoV-2 nsp3-4 pore complex

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Supramolecule #1: SARS-CoV-2 nsp3-4 pore complex

SupramoleculeName: SARS-CoV-2 nsp3-4 pore complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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 stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
10.0 mMTris-HClTris hydrochloride
1.0 mMEDTAEthylenediaminetetraacetic acid

Details: 150mM NaCl, 10mM Tris-HCl, 1mM EDTA
GridModel: EMS Lacey Carbon / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 3.0 e/Å2
Details: The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51 ...Details: The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51 to 51 (or -60 to 60) at 3 increments and an exposure dose of 3 e-/A2 per image. The total dose was 105 (or 123) e-/A2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFalcon 4i Selectris X energy filter
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. v4) / Number subtomograms used: 21715
ExtractionNumber tomograms: 4746 / Number images used: 598699 / Reference model: EMD-11514 / Software - Name: emClarity (ver. v1.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. v4)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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