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- EMDB-21955: Cryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotaviru... -

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Basic information

Entry
Database: EMDB / ID: EMD-21955
TitleCryo-EM reconstruction of VP5*/VP8* assembly from rhesus rotavirus particles - Upright conformation
Map data
SampleRotavirus VP4, VP6, VP7 assembly in the spike conformation:
(Outer capsid ...) x 2 / Intermediate capsid protein VP6 / (ligand) x 2
Function / homology
Function and homology information


viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / viral entry via permeabilization of inner membrane / receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment ...viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / viral entry via permeabilization of inner membrane / receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / structural molecule activity / membrane / metal ion binding
Glycoprotein VP7, domain 2 / Viral capsid/haemagglutinin protein / Glycoprotein VP7 / Rotavirus A/C, major capsid protein VP6 / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Concanavalin A-like lectin/glucanase domain superfamily / Outer capsid protein VP4 / Virus capsid protein, alpha-helical / Rotavirus VP4, membrane interaction domain / Rotavirus VP4 helical domain ...Glycoprotein VP7, domain 2 / Viral capsid/haemagglutinin protein / Glycoprotein VP7 / Rotavirus A/C, major capsid protein VP6 / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Concanavalin A-like lectin/glucanase domain superfamily / Outer capsid protein VP4 / Virus capsid protein, alpha-helical / Rotavirus VP4, membrane interaction domain / Rotavirus VP4 helical domain / Rotavirus VP4, membrane interaction domain superfamily / Glycoprotein VP7, domain 1
Intermediate capsid protein VP6 / Outer capsid protein VP4 / Outer capsid glycoprotein VP7
Biological speciesRotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3]) / RV-A (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHerrmann T / Harrison SC / Jenni S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)CA-13202 United States
CitationJournal: Nature / Year: 2021
Title: Functional refolding of the penetration protein on a non-enveloped virus.
Authors: Tobias Herrmann / Raúl Torres / Eric N Salgado / Cristina Berciu / Daniel Stoddard / Daniela Nicastro / Simon Jenni / Stephen C Harrison /
Abstract: A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4. Here we ...A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 10, 2020-
Header (metadata) releaseJan 20, 2021-
Map releaseJan 20, 2021-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wxe
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6wxe
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21955.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.)
1.23 Å/pix.
x 320 pix.
= 393.92 Å
1.23 Å/pix.
x 320 pix.
= 393.92 Å
1.23 Å/pix.
x 320 pix.
= 393.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.231 Å
Density
Contour LevelBy EMDB: 0.02 / Movie #1: 0.025
Minimum - Maximum-0.086855724 - 0.13865738
Average (Standard dev.)-7.4955324e-07 (±0.009578195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 393.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2311.2311.231
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z393.920393.920393.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0870.139-0.000

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Supplemental data

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Segmentation: #1

Fileemd_21955_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map 1

Fileemd_21955_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map 2

Fileemd_21955_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Rotavirus VP4, VP6, VP7 assembly in the spike conformation

EntireName: Rotavirus VP4, VP6, VP7 assembly in the spike conformation
Details: Obtained from wild-type recoated rhesus rotavirus particles (wt rcTLPs)
Number of components: 6

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Component #1: protein, Rotavirus VP4, VP6, VP7 assembly in the spike conformation

ProteinName: Rotavirus VP4, VP6, VP7 assembly in the spike conformation
Details: Obtained from wild-type recoated rhesus rotavirus particles (wt rcTLPs)
Recombinant expression: No
MassTheoretical: 1.7 MDa
SourceSpecies: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])

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Component #2: protein, Outer capsid protein VP4

ProteinName: Outer capsid protein VP4 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 86.655586 kDa
SourceSpecies: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Intermediate capsid protein VP6

ProteinName: Intermediate capsid protein VP6 / Number of Copies: 18 / Recombinant expression: No
MassTheoretical: 44.934766 kDa
SourceSpecies: RV-A (virus) / Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]

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Component #4: protein, Outer capsid glycoprotein VP7

ProteinName: Outer capsid glycoprotein VP7 / Number of Copies: 18 / Recombinant expression: No
MassTheoretical: 37.136531 kDa
SourceSpecies: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: ligand, 2-acetamido-2-deoxy-beta-D-glucopyranose

LigandName: 2-acetamido-2-deoxy-beta-D-glucopyranose / Number of Copies: 18 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Component #6: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 54 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.2 mg/mL / pH: 8
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 298.15 K / Humidity: 90 % / Details: 4 ul sample volume, 4 sec blotting time.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 33 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 27500 X (nominal), 40605 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 3000.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4107

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 167147
3D reconstructionAlgorithm: FOURIER SPACE / Software: cisTEM / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Reconstruction final map after classification of VP4 sub-particles. VP4 sub-particles extracted from viral particles (60 VP4 sub-particles per viral particle)
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL / Details: phenix.real_space_refine
Input PDB model: 4V7Q
Output model

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