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- PDB-8p4k: Vaccinia Virus palisade layer A10 trimer -

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Basic information

Entry
Database: PDB / ID: 8p4k
TitleVaccinia Virus palisade layer A10 trimer
ComponentsCore protein OPG136
KeywordsVIRAL PROTEIN / Poxvirus / Vaccinia Virus / core / cryo-electron tomography / AlphaFold
Function / homologyPoxvirus P4A / Poxvirus P4A protein / virion component / structural molecule activity / Major core protein OPG136 precursor
Function and homology information
Biological speciesVaccinia virus Western Reserve
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDatler, J. / Hansen, J.M. / Thader, A. / Schloegl, A. / Hodirnau, V.V. / Schur, F.K.M.
Funding support1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 31, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Core protein OPG136
B: Core protein OPG136
C: Core protein OPG136


Theoretical massNumber of molelcules
Total (without water)207,2053
Polymers207,2053
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Core protein OPG136 / 62 kDa peptide


Mass: 69068.242 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus Western Reserve / References: UniProt: P16715
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vaccinia virus Western Reserve / Type: VIRUS
Details: Purified from HeLa cells infected with Vaccinia Virus Western Reserve.
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Vaccinia virus Western Reserve
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 9
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.
Buffer component
IDConc.NameFormulaBuffer-ID
1210 mMPotassium chlorideKCl1
21 mMTris hydrochlorideTris-HCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Isolated viral cores with some detached soluble monodispersed particles.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K / Details: Leica GP2

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53.04 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 9264 / Details: 34 frames total.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selection
2EPU2.13image acquisition
4cryoSPARC4CTF correction
5RELION4CTF correction
8UCSF ChimeraX1.5model fittingrigid body fit
10cryoSPARC4initial Euler assignment
11RELION4final Euler assignment
12cryoSPARC4classification
13RELION4classification
14RELION43D reconstruction
15Rosetta3.13.20220812model refinementcentroid relax
Image processingDetails: dose weighted and motion corrected
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: OTHER / Num. of particles: 24943 / Details: Phenix FSCref following density modification / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Details: Rosetta relaxation
Atomic model buildingDetails: version 2.3 / Source name: AlphaFold / Type: in silico model

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