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- EMDB-24687: Cryo-EM structure of bluetongue virus capsid protein at low endos... -

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Basic information

Entry
Database: EMDB / ID: EMD-24687
TitleCryo-EM structure of bluetongue virus capsid protein at low endosomal pH intermediate state 3
Map dataIMS3
Sample
  • Virus: Bluetongue virus (serotype 1 / isolate South Africa)
Function / homologyOuter capsid protein VP5, Orbivirus / Orbivirus outer capsid protein VP5 / viral capsid / structural molecule activity / Outer capsid protein VP5
Function and homology information
Biological speciesBluetongue virus (serotype 1 / isolate South Africa)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsXia X / Wu WN / Cui YX / Roy P / Zhou ZH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386/DE028583/DE025567 United States
CitationJournal: Nat Microbiol / Year: 2021
Title: Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.
Authors: Xian Xia / Weining Wu / Yanxiang Cui / Polly Roy / Z Hong Zhou /
Abstract: Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration ...Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.
History
DepositionAug 13, 2021-
Header (metadata) releaseNov 3, 2021-
Map releaseNov 3, 2021-
UpdateNov 10, 2021-
Current statusNov 10, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24687.png

Downloads & links

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Map

FileDownload / File: emd_24687.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIMS3
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.08412507 - 0.136949
Average (Standard dev.)0.002548402 (±0.00963698)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 408.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z408.000408.000408.000
α/β/γ90.00090.00090.000
start NX/NY/NZ45440
NX/NY/NZ103105148
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0840.1370.003

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Supplemental data

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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
Details: The virus was generated from BHK-21 cell and purified by sucrose gradient.
NCBI-ID: 10905
Sci species name: Bluetongue virus (serotype 1 / isolate South Africa)
Sci species strain: BTV-1 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: sheep (sheep)
Virus shellShell ID: 1 / Diameter: 880.0 Å

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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

BufferpH: 5.5 / Component - Concentration: 20.0 mM / Component - Name: sodium citrate
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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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 magnification: 105000
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 K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3609 / Average exposure time: 8.0 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: 913440
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 120974

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Atomic model buiding 1

RefinementProtocol: OTHER

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