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- PDB-8coa: in situ Subtomogram average of Immature Rotavirus TLP spike -

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Basic information

Entry
Database: PDB / ID: 8coa
Titlein situ Subtomogram average of Immature Rotavirus TLP spike
Components
  • Intermediate capsid protein VP6
  • Outer capsid glycoprotein VP7
  • Outer capsid protein VP4
KeywordsVIRAL PROTEIN / Rotavirus / STA
Function / homology
Function and homology information


viral intermediate capsid / host cell endoplasmic reticulum lumen / host cell rough endoplasmic reticulum / T=13 icosahedral viral capsid / permeabilization of host organelle membrane involved in viral entry into host cell / host cytoskeleton / viral outer capsid / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / fusion of virus membrane with host plasma membrane ...viral intermediate capsid / host cell endoplasmic reticulum lumen / host cell rough endoplasmic reticulum / T=13 icosahedral viral capsid / permeabilization of host organelle membrane involved in viral entry into host cell / host cytoskeleton / viral outer capsid / 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 / metal ion binding / membrane
Similarity search - Function
Rotavirus A/C, major capsid protein VP6 / Rotavirus major capsid protein VP6 / Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Virus capsid protein, alpha-helical / Rotavirus VP4 helical domain / Rotavirus VP4 helical domain / Outer capsid protein VP4 ...Rotavirus A/C, major capsid protein VP6 / Rotavirus major capsid protein VP6 / Glycoprotein VP7 / Glycoprotein VP7, domain 1 / Glycoprotein VP7, domain 2 / Glycoprotein VP7 / Virus capsid protein, alpha-helical / Rotavirus VP4 helical domain / Rotavirus VP4 helical domain / Outer capsid protein VP4 / Rotavirus VP4, membrane interaction domain superfamily / Rotavirus VP4, membrane interaction domain / Rotavirus VP4 membrane interaction domain / Haemagglutinin outer capsid protein VP4, concanavalin-like domain / Outer Capsid protein VP4 (Hemagglutinin) Concanavalin-like domain / Viral capsid/haemagglutinin protein / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Outer capsid protein VP4 / Outer capsid glycoprotein VP7 / Intermediate capsid protein VP6
Similarity search - Component
Biological speciesRotavirus A
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 4.5 Å
AuthorsShah, P.N.M. / Stuart, D.I.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CAMS Innovation Fund for Medical Sciences (CIFMS)2018-I2M-2-002 United Kingdom
Wellcome Trust03141/Z/16/Z United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
CitationJournal: Cell Host Microbe / Year: 2023
Title: Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography.
Authors: Pranav N M Shah / James B Gilchrist / Björn O Forsberg / Alister Burt / Andrew Howe / Shyamal Mosalaganti / William Wan / Julika Radecke / Yuriy Chaban / Geoff Sutton / David I Stuart / Mark Boyce /
Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and ...Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.
History
DepositionFeb 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Apr 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer capsid protein VP4
B: Outer capsid protein VP4
C: Outer capsid protein VP4
D: Outer capsid glycoprotein VP7
E: Outer capsid glycoprotein VP7
F: Outer capsid glycoprotein VP7
G: Outer capsid glycoprotein VP7
H: Outer capsid glycoprotein VP7
I: Outer capsid glycoprotein VP7
J: Outer capsid glycoprotein VP7
K: Outer capsid glycoprotein VP7
L: Outer capsid glycoprotein VP7
M: Outer capsid glycoprotein VP7
N: Outer capsid glycoprotein VP7
O: Outer capsid glycoprotein VP7
P: Outer capsid glycoprotein VP7
d: Intermediate capsid protein VP6
e: Intermediate capsid protein VP6
f: Intermediate capsid protein VP6
g: Intermediate capsid protein VP6
h: Intermediate capsid protein VP6
i: Intermediate capsid protein VP6
j: Intermediate capsid protein VP6
k: Intermediate capsid protein VP6
l: Intermediate capsid protein VP6
m: Intermediate capsid protein VP6
n: Intermediate capsid protein VP6
o: Intermediate capsid protein VP6
p: Intermediate capsid protein VP6


Theoretical massNumber of molelcules
Total (without water)1,328,14229
Polymers1,328,14229
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer capsid protein VP4


Mass: 86767.953 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A / Gene: VP4 / Production host: Chlorocebus aethiops aethiops (mammal) / References: UniProt: A0A1Q2TSK9
#2: Protein
Outer capsid glycoprotein VP7


Mass: 37230.664 Da / Num. of mol.: 13
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A / Gene: VP7 / Production host: Chlorocebus aethiops aethiops (mammal) / References: UniProt: A0A1Q2TSM6
#3: Protein
Intermediate capsid protein VP6


Mass: 44910.738 Da / Num. of mol.: 13
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rotavirus A / Gene: VP6 / Production host: Chlorocebus aethiops aethiops (mammal) / References: UniProt: A2T3S6
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Rotavirus A / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Rotavirus A
Source (recombinant)Organism: Chlorocebus aethiops aethiops (mammal)
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MA104 cells infected with Rotavirus
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 2.19 e/Å2 / Avg electron dose per subtomogram: 90.2 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLOvolume selection
4Warp1.0.9CTF correction
7PHENIX1.2model fitting
10RELION4initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13Warp1.0.93D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16740 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 85 / Num. of volumes extracted: 16740
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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