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- EMDB-17414: Cryo-electron tomogram of isolated Vaccinia Virus cores -

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Basic information

Entry
Database: EMDB / ID: EMD-17414
TitleCryo-electron tomogram of isolated Vaccinia Virus cores
Map dataIsoNet corrected cryo-electron tomogram of isolated Vaccinia Virus cores
Sample
  • Virus: Vaccinia virus Western Reserve
KeywordsPoxvirus / Vaccinia Virus / core / cryo-electron tomography / AlphaFold / VIRAL PROTEIN
Biological speciesVaccinia virus Western Reserve
Methodelectron tomography / cryo EM
AuthorsDatler J / Hansen JM / Thader A / Schloegl A / Hodirnau VV / Schur FKM
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores.
Authors: Julia Datler / Jesse M Hansen / Andreas Thader / Alois Schlögl / Lukas W Bauer / Victor-Valentin Hodirnau / Florian K M Schur /
Abstract: Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural ...Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.
History
DepositionMay 22, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17414.png

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Map

FileDownload / File: emd_17414.map.gz / Format: CCP4 / Size: 162.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIsoNet corrected cryo-electron tomogram of isolated Vaccinia Virus cores
Voxel sizeX=Y=Z: 11.048 Å
Density
Minimum - Maximum-7.8389125 - 2.6801245
Average (Standard dev.)-0.3979616 (±0.28617632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600380187
Spacing380600187
CellA: 4198.24 Å / B: 6628.8003 Å / C: 2065.976 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 Western Reserve

EntireName: Vaccinia virus Western Reserve
Components
  • Virus: Vaccinia virus Western Reserve

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Supramolecule #1: Vaccinia virus Western Reserve

SupramoleculeName: Vaccinia virus Western Reserve / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Purified viral cores from HeLa cells infected with Vaccinia Virus Western Reserve.
NCBI-ID: 696871 / Sci species name: Vaccinia virus Western Reserve / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No

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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: 9 / Component - Concentration: 1.0 mM / Component - Formula: Tris-HClTris / Component - Name: Tris hydrochloride
Details: diluted 1:1 with 0.25% Trypsin (final concentration 0.125%) and 1:1 4% paraformaldehyde (final concentration 2%) in 1 mM Tris-HCl.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: LEICA EM GP / Details: Leica GP2.
Detailsintact and disrupted viral cores
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion Immuno Gold Reagents / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.361 µm / Nominal defocus min: 2.361 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsCryo-electron tomography data was collected with the SerialEM software package version 3.8 (Mastronarde 2005). New gain reference images were collected before data acquisition. DigitalMicrograph 3.4.3 as integrated into the Gatan Microscopy Suite v3.3 (Gatan) was used for filter tuning and SerialEM for microscope tuning.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 158.0 e/Å2 / Details: Images were collected in movie-mode with 10 frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Software - details: IsoNET 0.2 / Details: Tomogram was cropped and IsoNET corrected / Number images used: 43

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