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- PDB-5ow6: CryoEM structure of recombinant CMV particles with Tetanus-epitope -

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Basic information

Entry
Database: PDB / ID: 5ow6
TitleCryoEM structure of recombinant CMV particles with Tetanus-epitope
Components
  • Capsid protein, VP2
  • Capsid protein, VP3
  • VP1
KeywordsVIRUS LIKE PARTICLE / VLP / Vaccines / CryoEM / CMV
Function / homology
Function and homology information


T=3 icosahedral viral capsid / viral nucleocapsid / ribonucleoprotein complex / structural molecule activity / RNA binding
Similarity search - Function
Cucumovirus coat protein / Cucumovirus coat protein, subunit A superfamily / Cucumovirus coat protein
Similarity search - Domain/homology
Capsid protein / Capsid protein / Capsid protein
Similarity search - Component
Biological speciesCucumber mosaic virus (cucumber mosaic cucumovirus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKotecha, A. / Stuart, D.I. / Backmann, M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)G1000099 United Kingdom
Medical Research Council (United Kingdom)G1100525/1 United Kingdom
CitationJournal: NPJ Vaccines / Year: 2017
Title: Incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, Alzheimer's and cat allergy.
Authors: Andris Zeltins / Jonathan West / Franziska Zabel / Aadil El Turabi / Ina Balke / Stefanie Haas / Melanie Maudrich / Federico Storni / Paul Engeroff / Gary T Jennings / Abhay Kotecha / David ...Authors: Andris Zeltins / Jonathan West / Franziska Zabel / Aadil El Turabi / Ina Balke / Stefanie Haas / Melanie Maudrich / Federico Storni / Paul Engeroff / Gary T Jennings / Abhay Kotecha / David I Stuart / John Foerster / Martin F Bachmann /
Abstract: Monoclonal antibodies are widely used to treat non-infectious conditions but are costly. Vaccines could offer a cost-effective alternative but have been limited by sub-optimal T-cell stimulation ...Monoclonal antibodies are widely used to treat non-infectious conditions but are costly. Vaccines could offer a cost-effective alternative but have been limited by sub-optimal T-cell stimulation and/or weak vaccine responses in recipients, for example, in elderly patients. We have previously shown that the repetitive structure of virus-like-particles (VLPs) can effectively bypass self-tolerance in therapeutic vaccines. Their efficacy could be increased even further by the incorporation of an epitope stimulating T cell help. However, the self-assembly and stability of VLPs from envelope monomer proteins is sensitive to geometry, rendering the incorporation of foreign epitopes difficult. We here show that it is possible to engineer VLPs derived from a non human-pathogenic plant virus to incorporate a powerful T-cell-stimulatory epitope derived from Tetanus toxoid. These VLPs (termed CMV) retain self-assembly as well as long-term stability. Since Th cell memory to Tetanus is near universal in humans, CMV-based vaccines can deliver robust antibody-responses even under limiting conditions. By way of proof of concept, we tested a range of such vaccines against chronic inflammatory conditions (model: psoriasis, antigen: interleukin-17), neurodegenerative (Alzheimer's, β-amyloid), and allergic disease (cat allergy, Fel-d1), respectively. Vaccine responses were uniformly strong, selective, efficient , observed even in old mice, and employing low vaccine doses. In addition, randomly ascertained human blood cells were reactive to CMV-VLPs, confirming recognition of the incorporated Tetanus epitope. The CMV-VLP platform is adaptable to almost any antigen and its features and performance are ideally suited for the design of vaccines delivering enhanced responsiveness in aging populations.
History
DepositionAug 30, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 13, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 3, 2018Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.3May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_struct_oper_list
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: VP1
B: Capsid protein, VP2
C: Capsid protein, VP3


Theoretical massNumber of molelcules
Total (without water)59,1323
Polymers59,1323
Non-polymers00
Water00
1
A: VP1
B: Capsid protein, VP2
C: Capsid protein, VP3
x 60


Theoretical massNumber of molelcules
Total (without water)3,547,908180
Polymers3,547,908180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: VP1
B: Capsid protein, VP2
C: Capsid protein, VP3
x 5


  • icosahedral pentamer
  • 296 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)295,65915
Polymers295,65915
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: VP1
B: Capsid protein, VP2
C: Capsid protein, VP3
x 6


  • icosahedral 23 hexamer
  • 355 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)354,79118
Polymers354,79118
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein VP1


Mass: 16924.420 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: PGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEYDKKLVSRLQIRVNPLPKFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQANNKLLYDLSAMRADIGDMRKYAVLVYSKDDALETDELVLHVDIEHQRIPTSGVLPV
Source: (gene. exp.) Cucumber mosaic virus (cucumber mosaic cucumovirus)
Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta / References: UniProt: A0A1Q2SR16, UniProt: P69466*PLUS
#2: Protein Capsid protein, VP2


Mass: 21068.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cucumber mosaic virus (cucumber mosaic cucumovirus)
Gene: CP / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta / References: UniProt: K4TZS9, UniProt: P69466*PLUS
#3: Protein Capsid protein, VP3


Mass: 21139.229 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cucumber mosaic virus (cucumber mosaic cucumovirus)
Gene: CP / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta / References: UniProt: K4TZS9, UniProt: P69466*PLUS

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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: CryoEM structure of recombinant CMV particles with Tetanus-epitope
Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: cucumber mosaic cucumovirus (cucumber mosaic cucumovirus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Cucumis sativus
Virus shellTriangulation number (T number): 1
Buffer solutionpH: 9 / Details: 5 mM sodium borate, 2 mM EDTA, pH 9
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 5 mM sodium borate, 2 mM EDTA, pH 9
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C Flats 2/1 2C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 160000 X / Calibrated magnification: 37037 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 20 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 500
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 25 / Used frames/image: 2-25

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Processing

SoftwareName: PHENIX / Version: dev_2645: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN2particle selection
2SerialEM6.1image acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6600
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3582 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 174 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0064248
ELECTRON MICROSCOPYf_angle_d1.0095780
ELECTRON MICROSCOPYf_dihedral_angle_d4.9022609
ELECTRON MICROSCOPYf_chiral_restr0.058676
ELECTRON MICROSCOPYf_plane_restr0.007738

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