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- EMDB-43722: Cryo-EM structure of pre-subcore from in vitro assembled particles -

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Basic information

Entry
Database: EMDB / ID: EMD-43722
TitleCryo-EM structure of pre-subcore from in vitro assembled particles
Map data
Sample
  • Virus: Bluetongue virus (serotype 1 / isolate South Africa)
    • Protein or peptide: inner capsid protein VP3
    • Protein or peptide: inner capsid protein VP6
KeywordsBluetongue virus / capsid protein / assembly intermediate / virial proteins / VIRUS LIKE PARTICLE
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 / VP6 / Core protein VP3
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 15, 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_43722.png

Downloads & links

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Map

FileDownload / File: emd_43722.map.gz / Format: CCP4 / Size: 125 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 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.0013480434 - 1.9717581
Average (Standard dev.)0.015472153 (±0.09898997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43722_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_43722_half_map_2.map
Projections & Slices
AxesZYX

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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)
    • Protein or peptide: inner capsid protein VP3
    • Protein or peptide: inner capsid protein 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)

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Macromolecule #1: inner capsid protein VP3

MacromoleculeName: inner capsid protein VP3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILDD IKALAAEQVY KIVKVPSISF RHIVMQSRDR VLRVDTYYEE MSQVGDVITE DEPEKFYSTI IKKVRFIRGK GSFILHDIPT RDHRGMEVAE PEVLGVEFKN ...String:
MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILDD IKALAAEQVY KIVKVPSISF RHIVMQSRDR VLRVDTYYEE MSQVGDVITE DEPEKFYSTI IKKVRFIRGK GSFILHDIPT RDHRGMEVAE PEVLGVEFKN VLPVLTAEHR AMIQNALDGS IIENGNVATR DVDVFIGACS EPVYRIYNRL QGYIEAVQLQ ELRNSIGWLE RLGHRKRITY SQEVLTDFRR QDTIWVLALQ LPVNPQVVWD VPRSSIANLI MNIATCLPTG EYIAPNPRIS SITLTQRITT TGPFAILTGS TPTAQQLNDV RKIYLALMFP GQIILDLKID PGERMDPAVR MVAGVVGHLL FTAGGRFTNL TQNMARQLDI ALNDYLLYMY NTRVQVNYGP TGEPLDFQIG RNQYDCNVFR ADFATGTGYN GWATIDVEYR EPAPYVHAQR YIRYCGIDSR ELINPTTYGI GMTYHCYNEM LRMLVAAGKD SEAAYFRSML PFHMVRFARI NQIINEDLHS VFSLPDDMFN ALLPDLIAGA HQNADPVVLD VSWISLWFAF NRSFEPTHRN EMLEVAPLIE SVYASELSVM KVDMRHLSLM QRRFPDVLIQ ARPSHFWKAV LNDSPEAVKA VMNLSHSHNF INIRDMMRWV MLPSLQPSLK LALEEEAWAA ANDFEDLMLT DQVYMHRDML PEPRLDDIER FRQEGFYYTN MLEAPPEIDR VVQYTYEIAR LQANMGQFRA ALRRIMDDDD WVRFGGVLRT VRVKFYDARP PDDVLQGLPF SYDTNERGGL AYATIKYATE TTIFYLIYNV EFSNTPDSLV LINPTYTMTK VFINKRIVER VRVGQILAVL NRRFVAYKGK MRIMDITQSL KMGTKLAAPT V

UniProtKB: Core protein VP3

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Macromolecule #2: inner capsid protein VP6

MacromoleculeName: inner capsid protein VP6 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bluetongue virus (serotype 1 / isolate South Africa)
SequenceString: MSAAMLLAPG DVIKRSSEEL KQRQIQINLI DWTEGESEKE SKAEAKEGDK AEELKDGEGT QSESSQKKES SKETKDADVD RRIHTAVGSG SSAKGPGERA NENVDRGDGK VGGGGGDADA GVGATGANGG RWVVLTEEIA RAIESKYGTK IDVYRDEVPA QIIEVERSLQ ...String:
MSAAMLLAPG DVIKRSSEEL KQRQIQINLI DWTEGESEKE SKAEAKEGDK AEELKDGEGT QSESSQKKES SKETKDADVD RRIHTAVGSG SSAKGPGERA NENVDRGDGK VGGGGGDADA GVGATGANGG RWVVLTEEIA RAIESKYGTK IDVYRDEVPA QIIEVERSLQ KELGISREGV AEQTERLRDL RRKEKSGAHA KAAERGRRKQ GKKPHGDAQR EGTEEEKTSE EPASVGITIE GVMSQKKLLS MIGGVERKMA PIGARESAVM LVSNSIKDVV RATAYFTAPT GDPHWKEVAR EASKKKNILA YTSTGGDVKT EFLHLIDHL

UniProtKB: 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
DetailsIn vitro assembled capsid by using the purified proteins

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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: 9015 / 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: 64032
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 2 / Avg.num./class: 20000 / 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: C5 (5 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: 17277

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