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- EMDB-18918: Subtomogram average of the Vaccinia virus (WR) A4/A10 palisade tr... -

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Basic information

Entry
Database: EMDB / ID: EMD-18918
TitleSubtomogram average of the Vaccinia virus (WR) A4/A10 palisade trimer in mature virions
Map data
Sample
  • Virus: Vaccinia virus WR
    • Protein or peptide: Core protein A10
    • Protein or peptide: Core protein A4
KeywordsPalisade / A10 / A4 / p4a / Vaccinia / VIRAL PROTEIN
Function / homology
Function and homology information


virion component / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / structural molecule activity / membrane
Similarity search - Function
Orthopoxvirus A5 / Orthopoxvirus A5L protein-like / Poxvirus P4A / Poxvirus P4A protein
Similarity search - Domain/homology
Major core protein OPG136 precursor / 39kDa core protein OPG130
Similarity search - Component
Biological speciesVaccinia virus WR
Methodsubtomogram averaging / cryo EM / Resolution: 9.7 Å
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_18918.png

Downloads & links

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Map

FileDownload / File: emd_18918.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.56 Å
Density
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.1483242 - 0.1700079
Average (Standard dev.)-0.00016369166 (±0.00798003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 299.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18918_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18918_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18918_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 : Vaccinia virus WR

EntireName: Vaccinia virus WR
Components
  • Virus: Vaccinia virus WR
    • Protein or peptide: Core protein A10
    • Protein or peptide: Core protein A4

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Supramolecule #1: Vaccinia virus WR

SupramoleculeName: Vaccinia virus WR / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / 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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Macromolecule #1: Core protein A10

MacromoleculeName: Core protein A10 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus WR
Molecular weightTheoretical: 71.073477 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MMPIKSIVTL DQLEDSEYLF RIVSTVLPHL CLDYKVCDQL KTTFVHPFDI LLNNSLGSVT KQDELQAAIS KLGINYLIDT TSRELKLFN VTLNAGNIDI INTPINISSE TNPIINTHSF YDLPPFTQHL LNIRLTDTEY RARFIGGYIK PDGSDSMDVL A EKKYPDLN ...String:
MMPIKSIVTL DQLEDSEYLF RIVSTVLPHL CLDYKVCDQL KTTFVHPFDI LLNNSLGSVT KQDELQAAIS KLGINYLIDT TSRELKLFN VTLNAGNIDI INTPINISSE TNPIINTHSF YDLPPFTQHL LNIRLTDTEY RARFIGGYIK PDGSDSMDVL A EKKYPDLN FDNTYLFNIL YKDVINAPIK EFKAKIVNGV LSRQDFDNLI GVRQYITIQD RPRFDDAYNI ADAARHYGVN LN TLPLPNV DLTTMPTYKH LIMFEQYFIY TYDRVDIYYN GNKMLFDDEI INFTISMRYQ SLIPRLVDFF PDIPVNNNIV LHT RDPQNA AVNVTVALPN VQFVDINRNN KFFINFFNLL AKEQRSTAIK VTKSMFWDGM DYEEYKSKNL QDMMFINSTC YVFG LYNHN NTTYCSILSD IISAEKTPIR VCLLPRVVGG KTVTNLISET LKSISSMTIR EFPRKDKSIM HIGLSETGFM RFFQL LRLM ADKPHETAIK EVVMAYVGIK LGDKGSPYYI RKESYQDFIY LLFASMGFKV TTRRSIMGSN NISIISIRPR VTKQYI VAT LMKTSCSKNE AEKLITSAFD LLNFMVSVSD FRDYQSYRQY RNYCPRYFYA G

UniProtKB: Major core protein OPG136 precursor

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Macromolecule #2: Core protein A4

MacromoleculeName: Core protein A4 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Vaccinia virus WR
Molecular weightTheoretical: 30.95235 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDFFNKFSQG LAESSTPKSS IYYSEEKDPD TKKDEAIEIG LKSQESYYQR QLREQLARDN MTVASRQPIQ PLQPTIHITP QPVPTATPA PILLPSSTVP TPKPRQQTNT SSDMSNLFDW LSEDTDAPAS SLLPALTPSN AVQDIISKFN KDQKTTTPPS T QPSQTLPT ...String:
MDFFNKFSQG LAESSTPKSS IYYSEEKDPD TKKDEAIEIG LKSQESYYQR QLREQLARDN MTVASRQPIQ PLQPTIHITP QPVPTATPA PILLPSSTVP TPKPRQQTNT SSDMSNLFDW LSEDTDAPAS SLLPALTPSN AVQDIISKFN KDQKTTTPPS T QPSQTLPT TTCTQQSDGN ISCTTPTVTP PQPPIVATVC TPTPTGGTVC TTAQQNPNPG AASQQNLDDM ALKDLMSNVE RD MHQLQAE TNDLVTNVYD AREYTRRAID QILQLVKGFE RFQK

UniProtKB: 39kDa core protein OPG130

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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: 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

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Electron microscopy

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

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Image processing

ExtractionNumber tomograms: 98 / Number images used: 623174 / Software - Name: Dynamo
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number subtomograms used: 123492
FSC plot (resolution estimation)

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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
Output model

PDB-8r5i:
In situ structure of the Vaccinia virus (WR) A4/A10 palisade trimer in mature virions by flexible fitting into a cryoET map

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