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- EMDB-3855: CryoEM structure of recombinant CMV particles with Tetanus-epitope -

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Basic information

Entry
Database: EMDB / ID: EMD-3855
TitleCryoEM structure of recombinant CMV particles with Tetanus-epitope
Map data
SampleCryoEM structure of recombinant CMV particles with Tetanus-epitope != cucumber mosaic cucumovirus

CryoEM structure of recombinant CMV particles with Tetanus-epitope

  • Virus: cucumber mosaic cucumovirus (cucumber mosaic cucumovirus)
    • Protein or peptide: VP1
    • Protein or peptide: Capsid protein, VP2
    • Protein or peptide: Capsid protein, VP3
KeywordsVLP / Vaccines / CryoEM / CMV / VIRUS LIKE PARTICLE
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) / cucumber mosaic cucumovirus (cucumber mosaic cucumovirus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKotecha A / Stuart DI
Funding support United Kingdom, 2 items
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, 2017-
Header (metadata) releaseSep 13, 2017-
Map releaseSep 13, 2017-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ow6
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5ow6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3855.png

Downloads & links

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Map

FileDownload / File: emd_3855.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 400 pix.
= 540. Å		1.35 Å/pix.
x 400 pix.
= 540. Å		1.35 Å/pix.
x 400 pix.
= 540. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.05132204 - 0.12957124
Average (Standard dev.)0.00095981715 (±0.0073745414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 540.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z540.000540.000540.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0510.1300.001

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Supplemental data

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Sample components

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Entire : CryoEM structure of recombinant CMV particles with Tetanus-epitope

EntireName: CryoEM structure of recombinant CMV particles with Tetanus-epitope
Components
  • Virus: cucumber mosaic cucumovirus (cucumber mosaic cucumovirus)
    • Protein or peptide: VP1
    • Protein or peptide: Capsid protein, VP2
    • Protein or peptide: Capsid protein, VP3

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Supramolecule #1: cucumber mosaic cucumovirus

SupramoleculeName: cucumber mosaic cucumovirus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 12305 / Sci species name: cucumber mosaic cucumovirus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Cucumis sativus (cucumber)
Virus shellShell ID: 1 / T number (triangulation number): 1

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Macromolecule #1: VP1

MacromoleculeName: VP1 / type: protein_or_peptide / ID: 1
Details: PGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEYDKKLVSRLQIRVNPLPKFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQANNKLLYDLSAMRADIGDMRKYAVLVYSKDDALETDELVLHVDIEHQRIPTSGVLPV
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cucumber mosaic virus (cucumber mosaic cucumovirus)
Molecular weightTheoretical: 16.92442 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
PGYTFTSITL KPPKIDRGSY YGKRLLLPDS VTEYDKKLVS RLQIRVNPLP KFDSTVWVTV RKVPASSDLS VAAISAMFAD GASPVLVYQ YAASGVQANN KLLYDLSAMR ADIGDMRKYA VLVYSKDDAL ETDELVLHVD IEHQRIPTSG VLPV

UniProtKB: Capsid protein

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Macromolecule #2: Capsid protein, VP2

MacromoleculeName: Capsid protein, VP2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cucumber mosaic virus (cucumber mosaic cucumovirus)
Molecular weightTheoretical: 21.068152 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
DANFRVLSQQ LSRLNKTLAA GRPTINHPTF VGSERCRPGY TFTSITLKPP KIDRGSYYGK RLLLPDSVTE YDKKLVSRLQ IRVNPLPKF DSTVWVTVRK VPASSDLSVA AISAMFADGA SPVLVYQYAA SGVQANNKLL YDLSAMRADI GDMRKYAVLV Y SKDDALET DELVLHVDIE HQRIPTSGVL PV

UniProtKB: Capsid protein

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Macromolecule #3: Capsid protein, VP3

MacromoleculeName: Capsid protein, VP3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cucumber mosaic virus (cucumber mosaic cucumovirus)
Molecular weightTheoretical: 21.139229 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ADANFRVLSQ QLSRLNKTLA AGRPTINHPT FVGSERCRPG YTFTSITLKP PKIDRGSYYG KRLLLPDSVT EYDKKLVSRL QIRVNPLPK FDSTVWVTVR KVPASSDLSV AAISAMFADG ASPVLVYQYA ASGVQANNKL LYDLSAMRAD IGDMRKYAVL V YSKDDALE TDELVLHVDI EHQRIPTSGV LPV

UniProtKB: Capsid protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 9 / Details: 5 mM sodium borate, 2 mM EDTA, pH 9
GridModel: C Flats 2/1 2C / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 17 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details5 mM sodium borate, 2 mM EDTA, pH 9

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 2-25 / Number grids imaged: 1 / Number real images: 500 / Average exposure time: 5.0 sec. / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 160000
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6600
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1f15

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 3582
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 174 / Target criteria: Cross-correlation coefficient
Output model

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

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