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- PDB-8w1i: Cryo-EM structure of BTV subcore -

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Basic information

Entry
Database: PDB / ID: 8w1i
TitleCryo-EM structure of BTV subcore
ComponentsCore protein VP3
KeywordsVIRAL PROTEIN / Bluetongue virus / capsid protein / assembly intermediate / virion
Function / homologyInner layer core protein VP3, Orbivirus / Orbivirus VP3 (T2) protein / Inner layer core protein VP3, Reovirus / structural molecule activity / Core protein VP3
Function and homology information
Biological speciesBluetongue virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsXia, X. / Sung, P.Y. / Martynowycz, M.W. / Gonen, T. / Roy, P. / Zhou, Z.H.
Funding support United States, United Kingdom, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
Wellcome TrustWT221749/20/Z United Kingdom
CitationJournal: Cell / Year: 2024
Title: RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.
Authors: Xian Xia / Po-Yu Sung / Michael W Martynowycz / Tamir Gonen / Polly Roy / Z Hong Zhou /
Abstract: Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques ...Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.
History
DepositionFeb 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Core protein VP3
B: Core protein VP3
D: Core protein VP3
E: Core protein VP3
F: Core protein VP3
G: Core protein VP3
H: Core protein VP3
I: Core protein VP3
J: Core protein VP3
K: Core protein VP3


Theoretical massNumber of molelcules
Total (without water)1,034,10510
Polymers1,034,10510
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 VP3 / Major inner capsid protein


Mass: 103410.508 Da / Num. of mol.: 10 / Source method: isolated from a natural source
Source: (natural) Bluetongue virus (serotype 1 / isolate South Africa)
References: UniProt: Q1AE73

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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: Bluetongue virus (serotype 1 / isolate South Africa) / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Ovis aries
Buffer solutionpH: 8.8
Buffer componentConc.: 20 mM / Name: Tris
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Virus isolated from infected BHK cells
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 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: 81000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 62928
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazv0.2.5particle selection
2SerialEM4image acquisition
4RELION3.1CTF correction
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2124
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2124 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 159 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00471380
ELECTRON MICROSCOPYf_angle_d0.77696965
ELECTRON MICROSCOPYf_dihedral_angle_d5.1529685
ELECTRON MICROSCOPYf_chiral_restr0.04910840
ELECTRON MICROSCOPYf_plane_restr0.00612590

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