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- EMDB-2576: OSU Rotavirus non-trypsinized Triple Layered Particle -

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Basic information

Entry
Database: EMDB / ID: EMD-2576
TitleOSU Rotavirus non-trypsinized Triple Layered Particle
Map data3DR of non-tripsinized OSU strain rotavirus TLP
Sample
  • Sample: Non-trypsinized Triple Layered Particle from OSU strain rotavirus
  • Virus: Simian rotavirus A/SA11
Keywordsvirus assembly / rotavirus / Proteolysis
Biological speciesSimian rotavirus A/SA11
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 16.2 Å
AuthorsRodriguez JM / Chichon FJ / Martin-Forero E / Gonzalez-Camacho F / Carrascosa JL / Caston JR / Luque D
CitationJournal: PLoS Pathog / Year: 2014
Title: New insights into rotavirus entry machinery: stabilization of rotavirus spike conformation is independent of trypsin cleavage.
Authors: Javier M Rodríguez / Francisco J Chichón / Esther Martín-Forero / Fernando González-Camacho / José L Carrascosa / José R Castón / Daniel Luque /
Abstract: The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. ...The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.
History
DepositionFeb 12, 2014-
Header (metadata) releaseMar 12, 2014-
Map releaseJun 11, 2014-
UpdateJun 11, 2014-
Current statusJun 11, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2576.jpg

Downloads & links

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Map

FileDownload / File: emd_2576.map.gz / Format: CCP4 / Size: 357.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3DR of non-tripsinized OSU strain rotavirus TLP
Voxel sizeX=Y=Z: 2.175 Å
Density
Contour LevelBy AUTHOR: 1.02 / Movie #1: 1.02
Minimum - Maximum-6.44282198 - 6.76762295
Average (Standard dev.)0.06202898 (±0.9840591)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions458458458
Spacing458458458
CellA=B=C: 996.14996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1752.1752.175
M x/y/z458458458
origin x/y/z0.0000.0000.000
length x/y/z996.150996.150996.150
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS458458458
D min/max/mean-6.4436.7680.062

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Supplemental data

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Sample components

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Entire : Non-trypsinized Triple Layered Particle from OSU strain rotavirus

EntireName: Non-trypsinized Triple Layered Particle from OSU strain rotavirus
Components
  • Sample: Non-trypsinized Triple Layered Particle from OSU strain rotavirus
  • Virus: Simian rotavirus A/SA11

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Supramolecule #1000: Non-trypsinized Triple Layered Particle from OSU strain rotavirus

SupramoleculeName: Non-trypsinized Triple Layered Particle from OSU strain rotavirus
type: sample / ID: 1000 / Oligomeric state: icosahedral / Number unique components: 1

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Supramolecule #1: Simian rotavirus A/SA11

SupramoleculeName: Simian rotavirus A/SA11 / type: virus / ID: 1 / NCBI-ID: 10923 / Sci species name: Simian rotavirus A/SA11 / Sci species strain: OSU / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Sus scrofa (pig) / synonym: VERTEBRATES

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: Tris 25 mM, Na2HPO3 0.7 mM, NaCl 136.9 mM, KCl 5.1 mM, Glucose 5.6 mM, MgCl2 1 mM, CaCl2 1 mM
StainingType: NEGATIVE
Details: Samples were applied to grids, blotted and plunged into liquid ethane
GridDetails: R 2/2 Quantifoil grids
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
DateFeb 1, 2012
Image recordingDigitization - Sampling interval: 15 µm / Number real images: 162 / Average electron dose: 10 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping & amplitude decay
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.2 Å / Resolution method: OTHER / Software - Name: Xmipp / Number images used: 3690

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