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- EMDB-43714: Cryo-EM structure of VP3-VP6 heterohexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-43714
TitleCryo-EM structure of VP3-VP6 heterohexamer
Map data
Sample
  • Virus: Bluetongue virus (serotype 1 / isolate South Africa)
    • Protein or peptide: Core protein VP3
    • Protein or peptide: VP6
KeywordsBluetongue virus / capsid protein / heterohexamer / VIRAL PROTEIN
Function / homologyVP6 blue-tongue virus inner capsid protein / Orbivirus helicase VP6 / Inner layer core protein VP3, Orbivirus / Orbivirus VP3 (T2) protein / Inner layer core protein VP3, Reovirus / viral capsid / structural molecule activity / Core protein VP3 / VP6
Function and homology information
Biological speciesBluetongue virus (serotype 1 / isolate South Africa)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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 14, 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_43714.png

Downloads & links

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Map

FileDownload / File: emd_43714.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.0008736747 - 2.1416876
Average (Standard dev.)0.0077990624 (±0.071071886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43714_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43714_half_map_2.map
Projections & Slices
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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)
    • Protein or peptide: Core protein VP3
    • Protein or peptide: VP6

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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 / Macromolecule list: all / NCBI-ID: 10905
Sci species name: Bluetongue virus (serotype 1 / isolate South Africa)
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Ovis aries (sheep)
Molecular weightTheoretical: 460 KDa

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Macromolecule #1: Core protein VP3

MacromoleculeName: Core protein VP3 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Strain: BTV1
Molecular weightTheoretical: 105.613969 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAHHHHHHSS GLEVLFQGPG MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILD DIKALAAEQV YKIVKVPSIS FRHIVMQSRD RVLRVDTYYE EMSQVGDVIT EDEPEKFYST IIKKVRFIRG K GSFILHDI ...String:
MAHHHHHHSS GLEVLFQGPG MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILD DIKALAAEQV YKIVKVPSIS FRHIVMQSRD RVLRVDTYYE EMSQVGDVIT EDEPEKFYST IIKKVRFIRG K GSFILHDI PTRDHRGMEV AEPEVLGVEF KNVLPVLTAE HRAMIQNALD GSIIENGNVA TRDVDVFIGA CSEPVYRIYN RL QGYIEAV QLQELRNSIG WLERLGHRKR ITYSQEVLTD FRRQDTIWVL ALQLPVNPQV VWDVPRSSIA NLIMNIATCL PTG EYIAPN PRISSITLTQ RITTTGPFAI LTGSTPTAQQ LNDVRKIYLA LMFPGQIILD LKIDPGERMD PAVRMVAGVV GHLL FTAGG RFTNLTQNMA RQLDIALNDY LLYMYNTRVQ VNYGPTGEPL DFQIGRNQYD CNVFRADFAT GTGYNGWATI DVEYR EPAP YVHAQRYIRY CGIDSRELIN PTTYGIGMTY HCYNEMLRML VAAGKDSEAA YFRSMLPFHM VRFARINQII NEDLHS VFS LPDDMFNALL PDLIAGAHQN ADPVVLDVSW ISLWFAFNRS FEPTHRNEML EVAPLIESVY ASELSVMKVD MRHLSLM QR RFPDVLIQAR PSHFWKAVLN DSPEAVKAVM NLSHSHNFIN IRDMMRWVML PSLQPSLKLA LEEEAWAAAN DFEDLMLT D QVYMHRDMLP EPRLDDIERF RQEGFYYTNM LEAPPEIDRV VQYTYEIARL QANMGQFRAA LRRIMDDDDW VRFGGVLRT VRVKFYDARP PDDVLQGLPF SYDTNERGGL AYATIKYATE TTIFYLIYNV EFSNTPDSLV LINPTYTMTK VFINKRIVER VRVGQILAV LNRRFVAYKG KMRIMDITQS LKMGTKLAAP TV

UniProtKB: Core protein VP3

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Macromolecule #2: VP6

MacromoleculeName: VP6 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Strain: BTV1
Molecular weightTheoretical: 22.5655 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSAAMLLAPG DVIKRSSEEL KQRQIQINLI DWTEGESEKE SKAEAKEGDK AEELKDGEGT QSERDLRRKE KSGAHAKAAE RGRRKQGKK PHGDAQREGT EEEKTSEEPA SVGITIEGVM SQKKLLSMIG GVERKMAPIG ARESAVMLVS NSIKDVVRAT A YFTAPTGD ...String:
MSAAMLLAPG DVIKRSSEEL KQRQIQINLI DWTEGESEKE SKAEAKEGDK AEELKDGEGT QSERDLRRKE KSGAHAKAAE RGRRKQGKK PHGDAQREGT EEEKTSEEPA SVGITIEGVM SQKKLLSMIG GVERKMAPIG ARESAVMLVS NSIKDVVRAT A YFTAPTGD PHWKEVAREA SKKKNILAYT STGGDVKTEF LHLIDHL

UniProtKB: VP6, VP6

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMTris
100.0 mMSodium ChlorideNaClSodium chloride
1.0 mMDTT
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
DetailsRecombinated VP3-VP6 complex purified from insect cell

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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: 2.6 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / 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 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2843 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1451665
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
Final 3D classificationNumber classes: 5 / Avg.num./class: 120000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 138115

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 107
Output model

PDB-8w12:
Cryo-EM structure of VP3-VP6 heterohexamer

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