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- PDB-8r5i: In situ structure of the Vaccinia virus (WR) A4/A10 palisade trim... -

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Basic information

Entry
Database: PDB / ID: 8r5i
TitleIn situ structure of the Vaccinia virus (WR) A4/A10 palisade trimer in mature virions by flexible fitting into a cryoET map
Components
  • Core protein A10
  • Core protein A4
KeywordsVIRAL PROTEIN / Palisade / A10 / A4 / p4a / Vaccinia
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
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 9.7 Å
AuthorsCalcraft, T. / Hernandez-Gonzalez, M. / Nans, A. / Rosenthal, P.B. / Way, M.
Funding support United Kingdom, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Core protein A10
B: Core protein A4
C: Core protein A10
D: Core protein A4
E: Core protein A10
F: Core protein A4


Theoretical massNumber of molelcules
Total (without water)306,0776
Polymers306,0776
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21A
32A
42A
53A
63A
74A
84A
95A
105A
116A
126A

NCS domain segments:

Auth asym-ID: A / Label asym-ID: A

Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth seq-IDLabel seq-ID
111METMETPHEPHE1 - 5941 - 594
211METMETPHEPHE1 - 5941 - 594
322METMETPHEPHE1 - 5941 - 594
422METMETPHEPHE1 - 5941 - 594
533SERSERGLNGLN24 - 6624 - 66
633SERSERGLNGLN24 - 6624 - 66
744SERSERGLNGLN24 - 6624 - 66
844SERSERGLNGLN24 - 6624 - 66
955METMETPHEPHE1 - 5941 - 594
1055METMETPHEPHE1 - 5941 - 594
1166SERSERGLNGLN24 - 6624 - 66
1266SERSERGLNGLN24 - 6624 - 66

NCS ensembles :
IDDetails
1Global NCS restraints between domains: 1 2
2Global NCS restraints between domains: 3 4
3Global NCS restraints between domains: 5 6
4Global NCS restraints between domains: 7 8
5Global NCS restraints between domains: 9 10
6Global NCS restraints between domains: 11 12

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-0.499913, 0.866075, -0.000483), (-0.866076, -0.499913, 7.2E-5), (-0.000179, 0.000454, 1)94.99974, 354.3244, -0.04779
3given(-0.500137, -0.865946, -0.000177), (0.865946, -0.500137, 0.000232), (-0.00029, -3.7E-5, 1)354.37625, 94.94508, 0.05425
4given(-0.498019, 0.867166, -0.000312), (-0.867166, -0.498019, 5.0E-5), (-0.000112, 0.000296, 1)94.62159, 354.23911, -0.00245
5given(-0.478766, -0.877941, 0.001575), (0.877892, -0.478757, -0.009875), (0.009424, -0.003345, 0.99995)351.53622, 91.60132, -1.33757
6given(-0.499949, 0.866055, -0.000292), (-0.866055, -0.499949, 0.000185), (1.4E-5, 0.000345, 1)94.97627, 354.30676, -0.04195
7given(-0.522848, 0.852391, 0.007669), (-0.852372, -0.522895, 0.006581), (0.00962, -0.003096, 0.999949)99.80284, 353.58572, -1.42869

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Components

#1: Protein Core protein A10 / 23K / Core protein A10 / 23 kDa protein


Mass: 71073.477 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus WR / Gene: OPG136, VACWR129, A10L / Production host: Homo sapiens (human) / References: UniProt: P16715
#2: Protein Core protein A4 / p39 / Core protein A4 / 39kDa core protein OPG130


Mass: 30952.350 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus WR / Gene: OPG130, VACWR123, A4L / Production host: Homo sapiens (human) / References: UniProt: P29191

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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 WR / Type: VIRUS / Details: A36-YdF / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vaccinia virus WR
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 45mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295 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 FIELDBright-field microscopy / Nominal defocus max: 5000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 2.4 e/Å2 / Avg electron dose per subtomogram: 98.6 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Dynamovolume selection
2FEI tomographyimage acquisition
4GctfCTF correction
5RELION4CTF correction
8ISOLDEmodel fitting
10REFMACmodel refinement
12RELION4final Euler assignment
14RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 9.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123492 / Algorithm: FOURIER SPACE / Symmetry type: POINT
EM volume selectionNum. of tomograms: 98 / Num. of volumes extracted: 623174
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 9.7→9.7 Å / Cor.coef. Fo:Fc: 0.781 / WRfactor Rwork: 0.46 / SU B: 724.519 / SU ML: 4.415 / Average fsc free: 0 / Average fsc overall: 0.8949 / Average fsc work: 0.8949
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.4604 5651 -
all0.46 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 424.406 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01215804
ELECTRON MICROSCOPYr_bond_other_d00.01615182
ELECTRON MICROSCOPYr_angle_refined_deg1.5471.64821402
ELECTRON MICROSCOPYr_angle_other_deg0.521.5734921
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.53251905
ELECTRON MICROSCOPYr_dihedral_angle_2_deg8.436593
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.147102862
ELECTRON MICROSCOPYr_dihedral_angle_6_deg16.33910750
ELECTRON MICROSCOPYr_chiral_restr0.0650.22448
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.0218252
ELECTRON MICROSCOPYr_gen_planes_other0.0010.023710
ELECTRON MICROSCOPYr_nbd_refined0.2180.23378
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1890.213932
ELECTRON MICROSCOPYr_nbtor_refined0.170.27744
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0760.28528
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.0820.2363
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0210.21
ELECTRON MICROSCOPYr_mcbond_it50.09141.6337638
ELECTRON MICROSCOPYr_mcbond_other50.09141.6337638
ELECTRON MICROSCOPYr_mcangle_it89.89774.7929537
ELECTRON MICROSCOPYr_mcangle_other89.89274.8019538
ELECTRON MICROSCOPYr_scbond_it39.29743.7398166
ELECTRON MICROSCOPYr_scbond_other39.29543.7468167
ELECTRON MICROSCOPYr_scangle_it78.479.83611865
ELECTRON MICROSCOPYr_scangle_other78.39779.84211866
ELECTRON MICROSCOPYr_lrange_it194.4469.73665733
ELECTRON MICROSCOPYr_lrange_other194.399469.74165734
Refine LS restraints NCS

Auth asym-ID: A / Refine-ID: ELECTRON MICROSCOPY / Type: tight positional; tight thermal / Weight position: 0.07071

Ens-IDDom-IDRms dev Biso (Å2)Rms dev position (Å)Weight Biso
119.584690.074050.70709
129.584690.074050.70709
239.636120.069410.70709
249.636120.069410.70709
3510.019960.044920.70711
3610.019960.044920.70711
4713.470370.018160.70711
4813.470370.018160.70711
5910.774390.087610.70709
51010.774390.087610.70709
61116.666810.047690.70711
61216.666810.047690.70711
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: 0 / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
9.705-9.9561.3694421.3694420.7991.369
9.956-10.2280.5323950.5323950.8690.532
10.228-10.5230.3074320.3074320.9040.307
10.523-10.8450.2983590.2983590.9080.298
10.845-11.1990.2373760.2373760.9330.237
11.199-11.5890.223790.223790.9390.22
11.589-12.0240.2373020.2373020.9380.237
12.024-12.5120.2693490.2693490.9260.269
12.512-13.0640.3023400.3023400.9160.302
13.064-13.6960.3492810.3492810.9120.349
13.696-14.4310.4172860.4172860.9080.417
14.431-15.2970.4482750.4482750.9080.448
15.297-16.3410.4912620.4912620.9210.491
16.341-17.6330.5142370.5142370.9260.514
17.633-19.2890.5512090.5512090.8670.551
19.289-21.5210.5252190.5252190.8520.525
21.521-24.7660.4121740.4121740.8040.412
24.766-30.1260.3541440.3541440.7610.354
30.126-41.7680.5381210.5381210.8990.538
41.768-149.760.783690.783690.9710.783

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