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- PDB-7bjp: The cryo-EM structure of vesivirus 2117, an adventitious agent an... -

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Entry
Database: PDB / ID: 7bjp
TitleThe cryo-EM structure of vesivirus 2117, an adventitious agent and possible cause of haemorrhagic gastroenteritis in dogs.
ComponentsCapsid proteinCapsid
KeywordsVIRAL PROTEIN / capsid / calicivirus / vp1
Function / homologyCalicivirus coat protein / Calicivirus coat protein / T=3 icosahedral viral capsid / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / host cell cytoplasm / cytoplasm / Capsid protein
Function and homology information
Biological speciesCalicivirus isolate 2117
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsSutherland, H. / Conley, M.J. / Emmott, E. / Streetley, J. / Goodfellow, I.G. / Bhella, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_12014/7 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T002239/1 United Kingdom
Citation
Journal: J Virol / Year: 2021
Title: The Cryo-EM Structure of Vesivirus 2117 Highlights Functional Variations in Entry Pathways for Viruses in Different Clades of the Genus.
Authors: Hazel Sutherland / Michaela J Conley / Edward Emmott / James Streetley / Ian G Goodfellow / David Bhella /
Abstract: Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial ...Vesivirus 2117 is an adventitious agent that has been responsible for lost productivity in biopharmaceutical production following contamination of Chinese hamster ovary cell cultures in commercial bioreactors. A member of the , 2117 is classified within the Vesivirus genus in a clade that includes canine and mink caliciviruses but is distinct from the vesicular exanthema of swine virus (VESV) clade, which includes the extensively studied feline calicivirus (FCV). We have used cryogenic electron microscopy (cryo-EM) to determine the structure of the capsid of this small, icosahedral, positive-sense-RNA-containing virus. We show that the outer face of the dimeric capsomeres, which contains the receptor binding site and major immunodominant epitopes in all caliciviruses studied thus far, is quite different from that of FCV. This is a consequence of a 22-amino-acid insertion in the sequence of the FCV major capsid protein that forms a "cantilevered arm" that both plays an important role in receptor engagement and undergoes structural rearrangements thought to be important for genome delivery to the cytosol. Our data highlight a potentially important difference in the attachment and entry pathways employed by the different clades of the genus. Vesivirus 2117 has caused significant losses in manufacturing of biopharmaceutical products following contamination of cell cultures used in their production. We report the structure of the vesivirus 2117 capsid, the shell that encloses the virus's genome. Comparison of this structure with that of a related vesivirus, feline calicivirus (FCV), highlighted potentially important differences related to virus attachment and entry. Our findings suggest that these two viruses may bind differently to receptors at the host cell surface. We also show that a region of the capsid protein of FCV that rearranges following receptor engagement is not present in vesivirus 2117. These structural changes in the FCV capsid have been shown to allow the assembly of a portal-like structure that is hypothesized to deliver the viral genome to the cell's interior. Our data suggest that the 2117 portal assembly may employ a different means of anchoring to the outer face of the capsid.
#1: Journal: Biorxiv / Year: 2021
Title: The cryo-EM structure of vesivirus 2117 highlights functional variations in entry pathways for viruses in different clades of the Vesivirus genus
Authors: Sutherland, H. / Conley, M.J. / Emmott, E. / Streetley, J. / Goodfellow, I.G. / Bhella, D.
History
DepositionJan 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2021Group: Database references / Category: citation / citation_author
Revision 1.2Jun 23, 2021Group: Database references / Derived calculations / Category: citation / citation_author / struct_sheet
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _struct_sheet.number_strands

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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein


Theoretical massNumber of molelcules
Total (without water)175,2083
Polymers175,2083
Non-polymers00
Water0
1
A: Capsid protein
B: Capsid protein
C: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)10,512,508180
Polymers10,512,508180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
MethodUCSF CHIMERA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A27 - 526
2010B27 - 526
1020A12 - 527
2020C12 - 527
1030B27 - 526
2030C27 - 526

NCS ensembles :
ID
1
2
3

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Components

#1: Protein Capsid protein / Capsid / Coat protein


Mass: 58402.820 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Calicivirus isolate 2117 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q6VCZ3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Calicivirus isolate 2117 / Type: VIRUS
Details: The major capsid protein VP1 was expressed in a baculovirus system - in Hi5 cells
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Calicivirus isolate 2117
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: unidentified
Virus shellName: VP1 / Diameter: 400 nm / Triangulation number (T number): 3
Buffer solutionpH: 7.4 / Details: Phosphate buffered saline
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Virus-like particles
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: Sample was loaded onto C-flat C1.2/1.3 grids bearing a thin continuous carbon film, allowed to adsorb for one minute before blotting for 3 seconds and plunging into liquid ethane.

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 2 sec. / Electron dose: 55 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-64 (8k x 8k) / Num. of grids imaged: 5 / Num. of real images: 2000
Details: Data collection was performed as part of installation of the new CryoARM 300 microscope - five short sessions were run using primarily JADAS although one collection was performed using SerialEM.
Image scansWidth: 8192 / Height: 8192 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2JADASimage acquisition
4GctfCTF correction
5RELION3.0.3CTF correction
8Cootmodel fitting
10RELION3.0.3initial Euler assignment
11RELION3.0.3final Euler assignment
12RELION3.0.3classification
13RELION3.0.33D reconstruction
14PHENIXmodel refinement
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 65071
Details: Automated particle picking was performed using RELION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15989 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementResolution: 3.8→221.56 Å / Cor.coef. Fo:Fc: 0.478 / SU B: 141.154 / SU ML: 2.046 / ESU R: 0.481
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.54817 --
obs0.54817 270613 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 97.957 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20.22 Å20.14 Å2
2---0.81 Å20.19 Å2
3---0.84 Å2
Refinement stepCycle: 1 / Total: 11902
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01277706
ELECTRON MICROSCOPYf_angle_d0.986105915
ELECTRON MICROSCOPYf_dihedral_angle_d9.24710366
ELECTRON MICROSCOPYf_chiral_restr0.06311674
ELECTRON MICROSCOPYf_plane_restr0.00713609
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A241560.27
12B241560.27
21A242700.28
22C242700.28
31B242940.28
32C242940.28
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.543 20061 -
obs--100 %

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