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- PDB-8anw: Poliovirus type 3 (strain Saukett) stabilised virus-like particle... -

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Basic information

Entry
Database: PDB / ID: 8anw
TitlePoliovirus type 3 (strain Saukett) stabilised virus-like particle (PV3 SC8).
Components
  • Capsid protein, VP0
  • Capsid protein, VP1
  • Capsid protein, VP3
KeywordsVIRUS LIKE PARTICLE / Capsid protein
Function / homology
Function and homology information


symbiont genome entry into host cell via pore formation in plasma membrane / viral capsid / host cell cytoplasm / symbiont-mediated suppression of host gene expression / virion attachment to host cell / structural molecule activity
Similarity search - Function
Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Picornavirus capsid / picornavirus capsid protein / Picornavirus/Calicivirus coat protein / Viral coat protein subunit
Similarity search - Domain/homology
SPHINGOSINE / Genome polyprotein
Similarity search - Component
Biological speciesHuman poliovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBahar, M.W. / Fry, E.E. / Stuart, D.I.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationRG.IMCB.I8-TSA-083 United States
CitationJournal: Viruses / Year: 2022
Title: Production and Characterisation of Stabilised PV-3 Virus-like Particles Using .
Authors: Lee Sherry / Keith Grehan / Jessica J Swanson / Mohammad W Bahar / Claudine Porta / Elizabeth E Fry / David I Stuart / David J Rowlands / Nicola J Stonehouse /
Abstract: Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and ...Following the success of global vaccination programmes using the live-attenuated oral and inactivated poliovirus vaccines (OPV and IPV), wild poliovirus (PV) is now only endemic in Afghanistan and Pakistan. However, the continued use of these vaccines poses potential risks to the eradication of PV. The production of recombinant PV virus-like particles (VLPs), which lack the viral genome offer great potential as next-generation vaccines for the post-polio world. We have previously reported production of PV VLPs using , however, these VLPs were in the non-native conformation (C Ag), which would not produce effective protection against PV. Here, we build on this work and show that it is possible to produce wt PV-3 and thermally stabilised PV-3 (referred to as PV-3 SC8) VLPs in the native conformation (D Ag) using . We show that the PV-3 SC8 VLPs provide a much-improved D:C antigen ratio as compared to wt PV-3, whilst exhibiting greater thermostability than the current IPV vaccine. Finally, we determine the cryo-EM structure of the yeast-derived PV-3 SC8 VLPs and compare this to previously published PV-3 D Ag structures, highlighting the similarities between these recombinantly expressed VLPs and the infectious virus, further emphasising their potential as a next-generation vaccine candidate for PV.
History
DepositionAug 6, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _citation.page_first / _citation.pdbx_database_id_PubMed ..._citation.page_first / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Capsid protein, VP1
B: Capsid protein, VP0
C: Capsid protein, VP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,8004
Polymers97,5013
Non-polymers2991
Water00
1
A: Capsid protein, VP1
B: Capsid protein, VP0
C: Capsid protein, VP3
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)5,868,024240
Polymers5,850,054180
Non-polymers17,97060
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation59

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Components

#1: Protein Capsid protein, VP1


Mass: 33562.785 Da / Num. of mol.: 1 / Mutation: VP1 T105M, VP1 F132L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human poliovirus 3 / Cell line (production host): PichiaPink / Production host: Komagataella pastoris (fungus) / References: UniProt: Q84895
#2: Protein Capsid protein, VP0


Mass: 37623.023 Da / Num. of mol.: 1 / Mutation: VP2 L18I, VP2 L215M, VP2 D241E, VP4 T67A
Source method: isolated from a genetically manipulated source
Details: Sequence is given for the VP0 polypeptide. Mutations are numbered according to sequence numbering for mature polypeptides VP2 and VP4.
Source: (gene. exp.) Human poliovirus 3 / Cell line (production host): PichiaPink / Production host: Komagataella pastoris (fungus) / References: UniProt: Q84895
#3: Protein Capsid protein, VP3


Mass: 26315.100 Da / Num. of mol.: 1 / Mutation: VP3 H19Y, VP3 L85F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human poliovirus 3 / Cell line (production host): PichiaPink / Production host: Komagataella pastoris (fungus) / References: UniProt: Q84895
#4: Chemical ChemComp-SPH / SPHINGOSINE


Mass: 299.492 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H37NO2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Human poliovirus 3 / Type: VIRUS
Details: Recombinantly expressed virus-like particle of PV3 (strain Saukett).
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 5.85 MDa / Experimental value: NO
Source (natural)Organism: Human poliovirus 3 / Strain: Saukett
Source (recombinant)Organism: Komagataella pastoris (fungus) / Cell: PichiaPink
Details of virusEmpty: YES / Enveloped: NO / Isolate: SEROTYPE / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Virus shellName: Virus shell 1 / Diameter: 310 nm / Triangulation number (T number): 1
Buffer solutionpH: 7 / Details: 1 x DPBS, 20 mM EDTA, pH 7.0
Buffer component
IDConc.NameBuffer-ID
11 xDPBS1
220 mMEDTA1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Virus-like particle purified by sucrose density gradient purification from Pichia pastoris cells.
Specimen supportDetails: The specific type of grid used was Ultra-thin carbon support film, 3nm - on lacey carbon AGS187-4 from Agar Scientific.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Double blotting with 4 ul of sample, followed by 4 second blot, before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 60975 X / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3717 / Details: Pixel sampling of 0.82 A/pixel.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: (1.19.2_4158:phenix.real_space_refine) / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.5.6particle selectioncrYOLO was used to pick particles automatically
2SerialEMimage acquisition
4CTFFIND4CTF correctionCTFFIND4 in RELION was used for CTF correction
7UCSF Chimera1.14model fitting
9RELION3.1.1initial Euler assignment
10RELION3.1.1final Euler assignment
11RELION3.1.1classification
12RELION3.1.13D reconstruction
13PHENIX1.19.2-4158-000model refinement
Image processingDetails: Pixels size was 0.82 A/pixel
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10744
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2712 / Algorithm: BACK PROJECTION
Details: Final reconstruction was sharpened with Post-processing in RELION using an inverse B-factor of -69.1 Angstroms.
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Details: Initial model was rigid body fitted using UCSF chimera and Coot. Global minimization and B-factor refinement was performed in real space using phenix_real.space.refine.
Atomic model buildingPDB-ID: 6Z6W

6z6w


Accession code: 6Z6W / Source name: PDB / Type: experimental model
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 57.31 Å2 / Biso mean: 13.0392 Å2 / Biso min: 5.18 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00234854
ELECTRON MICROSCOPYf_angle_d0.48547514
ELECTRON MICROSCOPYf_chiral_restr0.0435214
ELECTRON MICROSCOPYf_plane_restr0.0046108
ELECTRON MICROSCOPYf_dihedral_angle_d11.58912702

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