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- EMDB-18916: Cryotomogram of mature Vaccinia virus (WR) virion -

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Basic information

Entry
Database: EMDB / ID: EMD-18916
TitleCryotomogram of mature Vaccinia virus (WR) virion
Map data
Sample
  • Virus: Vaccinia virus WR
KeywordsPalisade / Portal / Vaccinia / VIRAL PROTEIN
Biological speciesVaccinia virus WR
Methodelectron tomography / cryo EM
AuthorsCalcraft T / Hernandez-Gonzalez M / Nans A / Rosenthal PB / Way M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
The Francis Crick Institute United Kingdom
CitationJournal: mBio / Year: 2024
Title: Palisade structure in intact vaccinia virions.
Authors: Miguel Hernandez-Gonzalez / Thomas Calcraft / Andrea Nans / Peter B Rosenthal / Michael Way /
Abstract: Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a ...Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.
History
DepositionNov 16, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18916.png

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Map

FileDownload / File: emd_18916.map.gz / Format: CCP4 / Size: 2.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 6.24 Å
Density
Minimum - Maximum-0.6508105 - 0.29225957
Average (Standard dev.)0.023340514 (±0.014489076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-300
Dimensions10241024600
Spacing10241024600
CellA: 6389.76 Å / B: 6389.76 Å / C: 3743.9998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Vaccinia virus WR

EntireName: Vaccinia virus WR
Components
  • Virus: Vaccinia virus WR

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Supramolecule #1: Vaccinia virus WR

SupramoleculeName: Vaccinia virus WR / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: A36-YdF / NCBI-ID: 10254 / Sci species name: Vaccinia virus WR / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Details: 45mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI Solutions / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 2.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software: (Name: IMOD, Topaz)
Details: Tomogram was denoised using Topaz with the default pretrained model
Number images used: 41

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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