+Open data
-Basic information
Entry | Database: PDB / ID: 8coa | ||||||||||||||||||
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Title | in situ Subtomogram average of Immature Rotavirus TLP spike | ||||||||||||||||||
Components |
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Keywords | VIRAL 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 / host cytoskeleton / symbiont entry into host cell via permeabilization of inner membrane / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding ...viral intermediate capsid / host cell endoplasmic reticulum lumen / host cell rough endoplasmic reticulum / T=13 icosahedral viral capsid / host cytoskeleton / symbiont entry into host cell via permeabilization of inner membrane / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into 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 Similarity search - Function | ||||||||||||||||||
Biological species | Rotavirus A | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 4.5 Å | ||||||||||||||||||
Authors | Shah, P.N.M. / Stuart, D.I. | ||||||||||||||||||
Funding support | United Kingdom, 5items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8coa.cif.gz | 1.9 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8coa.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8coa.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8coa_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8coa_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 8coa_validation.xml.gz | 236.4 KB | Display | |
Data in CIF | 8coa_validation.cif.gz | 374.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/co/8coa ftp://data.pdbj.org/pub/pdb/validation_reports/co/8coa | HTTPS FTP |
-Related structure data
Related structure data | 16774MC 8bp8C 8co6C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 | 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 | 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 modification | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: CELL / 3D reconstruction method: subtomogram averaging |
-Sample preparation
Component | Name: Rotavirus A / Type: VIRUS / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Rotavirus A |
Source (recombinant) | Organism: Chlorocebus aethiops aethiops (mammal) |
Details of virus | Empty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MA104 cells infected with Rotavirus |
Specimen support | Grid material: COPPER / Grid type: Quantifoil R2/1 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm |
Image recording | Electron 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) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16740 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
EM volume selection | Num. of tomograms: 85 / Num. of volumes extracted: 16740 | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL |