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- PDB-8wd7: Structural organization of the palisade layer in isolated vaccini... -

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Basic information

Entry
Database: PDB / ID: 8wd7
TitleStructural organization of the palisade layer in isolated vaccinia virus cores
ComponentsCore protein OPG136
KeywordsVIRAL PROTEIN / poxvirus / assembly / core / palisade layer / A10
Function / homologyPoxvirus P4A / Poxvirus P4A protein / virion component / structural molecule activity / Major core protein OPG136 precursor
Function and homology information
Biological speciesVaccinia virus
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 10 Å
AuthorsLiu, Y. / Qu, X. / Duan, M. / Shi, X. / Liu, S. / Shi, Y. / Gao, G.F.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: To Be Published
Title: Cryo-ET reveals A10 protein as a major component of the poxvirus palisade layer
Authors: Liu, Y. / Qu, X. / Duan, M. / Shi, X. / Liu, S. / Shi, Y. / Gao, G.F.
History
DepositionSep 14, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release

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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)213,2203
Polymers213,2203
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Core protein OPG136 / 62 kDa peptide / Coordinate model: Cα atoms only


Mass: 71073.477 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus (strain Western Reserve) / Cell line: grown in HeLa cells / References: UniProt: P16715

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Vaccinia virus Western Reserve / Type: VIRUS
Details: Vaccinia virus was grown in HeLa cells and purified. The resultant mature virions were treated with DTT and NP40 and then purified to yield viral cores.
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Vaccinia virus Western Reserve
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 9 / Details: 1mM Tris pH 9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Vaccinia virus was grown in HeLa cells and purified. The resultant mature virions were treated with DTT and NP40 and then purified to yield viral cores. Isolated cores were used as a specimen.
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 53000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1600 nm / Cs: 0 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 2.9 e/Å2 / Avg electron dose per subtomogram: 119 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Details: Dose rate 5.2 e-/pixel/s, EER (241 frames/sec)
EM imaging opticsEnergyfilter name: TFS Selectris X
Chromatic aberration corrector: Microscope was equipped with a Cc corrector
Energyfilter slit width: 40 eV
Spherical aberration corrector: Microscope was equipped with a Cs corrector

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Processing

EM software
IDNameVersionCategoryDetails
1Dynamovolume selection
2SerialEM3.9image acquisition
4Warp1.1.0CTF correction
8UCSF Chimeramodel fittingRigid body fitting
12RELIONfinal Euler assignment
13Dynamofinal Euler assignment
15RELION3.133D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 10 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207056 / Algorithm: FOURIER SPACE / Symmetry type: POINT
EM volume selectionDetails: Tomograms generated in Warp were denoised and corrected for missing wedge information using IsoNet. Subtomograms were selected by oversampling points at 4 nm distance using a surface model ...Details: Tomograms generated in Warp were denoised and corrected for missing wedge information using IsoNet. Subtomograms were selected by oversampling points at 4 nm distance using a surface model in Dynamo. In essence, for each viral core, a 3D surface was generated by fitting manually selected points on the core surface.
Num. of tomograms: 74 / Num. of volumes extracted: 572775
Atomic model buildingSource name: AlphaFold / Type: in silico model

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