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- EMDB-43728: Subtomogram averaging of BTV virion in host cells -

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Basic information

Entry
Database: EMDB / ID: EMD-43728
TitleSubtomogram averaging of BTV virion in host cells
Map data
Sample
  • Virus: Bluetongue virus (serotype 1 / isolate South Africa)
KeywordsBluetongue virus / capsid protein / assembly intermediate / virion / VIRUS
Biological speciesBluetongue virus (serotype 1 / isolate South Africa)
Methodsubtomogram averaging / cryo EM / Resolution: 7.2 Å
AuthorsXia X / Sung PY / Martynowycz MW / Gonen T / Roy P / Zhou ZH
Funding support United States, United Kingdom, 2 items
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, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43728.png

Downloads & links

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Map

FileDownload / File: emd_43728.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.61 Å/pix.
x 200 pix.
= 521. Å		2.61 Å/pix.
x 200 pix.
= 521. Å		2.61 Å/pix.
x 200 pix.
= 521. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.605 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.078
Minimum - Maximum-0.29666123 - 0.40760678
Average (Standard dev.)0.000000000000285 (±0.04840882)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 521.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43728_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_43728_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 : Bluetongue virus (serotype 1 / isolate South Africa)

EntireName: Bluetongue virus (serotype 1 / isolate South Africa)
Components
  • Virus: Bluetongue virus (serotype 1 / isolate South Africa)

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Supramolecule #1: Bluetongue virus (serotype 1 / isolate South Africa)

SupramoleculeName: Bluetongue virus (serotype 1 / isolate South Africa) / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 10905
Sci species name: Bluetongue virus (serotype 1 / isolate South Africa)
Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Ovis aries (sheep)
Virus shellShell ID: 1 / Name: icosahedral / Diameter: 880.0 Å

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 33000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 5 / Average exposure time: 0.3 sec. / Average electron dose: 3.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 14 / Number images used: 7112 / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 3500 / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number subtomograms used: 3562

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