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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
DescriptorVP1, Capsid protein, VP2, Capsid protein, VP3
KeywordsVIRUS LIKE PARTICLE / VLP / Vaccines / CryoEM / CMV
Specimen sourceCucumber mosaic virus / virus / cucumber mosaic cucumovirus
MethodElectron microscopy (4.2 Å resolution / Particle / Single particle)
AuthorsKotecha, A. / Stuart, D.I. / Backmann, M.
Citationto be published

to be published Search PubMed
Incorporation of Tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, Alzheimers and cat allergy.
Zeltins, A. / West, J. / Zabel, F. / El-Turabi, A. / Jennings, A. / Balke, I. / Kotecha, A. / Stuart, D.I. / Storni, F. / Engeroff, P. / Maudrich, M. / Haas, S. / Bachmann, M. / Foerster, J.

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 30, 2017 / Release: Sep 13, 2017

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Structure visualization

Movie
  • 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
  • EMDB-3855
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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
Polyers59,1323
Non-polymers00
Water0
#1
A: VP1
B: Capsid protein, VP2
C: Capsid protein, VP3
x 60


Theoretical massNumber of molelcules
Total (without water)3,547,908180
Polyers3,547,908180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
#2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
#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
Polyers295,65915
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
#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
Polyers354,79118
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
#5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Polypeptide(L)VP1


Mass: 16924.420 Da / Num. of mol.: 1
Details: PGYTFTSITLKPPKIDRGSYYGKRLLLPDSVTEYDKKLVSRLQIRVNPLP KFDSTVWVTVRKVPASSDLSVAAISAMFADGASPVLVYQYAASGVQANNK LLYDLSAMRADIGDMRKYAVLVYSKDDALETDELVLHVDIEHQRIPTSGV LPV
Source: (gene. exp.) Cucumber mosaic virus / virus / References: UniProt: A0A1Q2SR16
#2: Polypeptide(L)Capsid protein, VP2


Mass: 21068.152 Da / Num. of mol.: 1 / Source: (gene. exp.) Cucumber mosaic virus / virus / References: UniProt: K4TZS9
#3: Polypeptide(L)Capsid protein, VP3


Mass: 21139.229 Da / Num. of mol.: 1 / Source: (gene. exp.) Cucumber mosaic virus / virus / References: UniProt: K4TZS9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: CryoEM structure of recombinant CMV particles with Tetanus-epitope
Type: VIRUS / Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: cucumber mosaic cucumovirus
Source (recombinant)Organism: Escherichia coli
Details of virusEmpty: YES / Enveloped: NO / Virus isolate: OTHER / Virus type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Cucumis sativus
Virus shellTriangulation number (T number): 1
Buffer solutionDetails: 5 mM sodium borate, 2 mM EDTA, pH 9 / pH: 9
SpecimenConc.: 2.5 mg/ml / Details: 5 mM sodium borate, 2 mM EDTA, pH 9 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: C Flats 2/1 2C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 160000 / Calibrated magnification: 37037 / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 50 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Temperature (max): 70 kelvins / Temperature (min): 70 kelvins
Image recordingAverage exposure time: 5 sec. / Electron dose: 20 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 500
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 microns / Dimension width: 3838 / Dimension 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
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1EMAN2PARTICLE SELECTION1
2SerialEM6.1IMAGE ACQUISITION1
4CTFFIND4CTF CORRECTION1
7UCSF ChimeraMODEL FITTING1
9RELION1.4INITIAL EULER ASSIGNMENT1
10RELION1.4FINAL EULER ASSIGNMENT1
11RELION1.4CLASSIFICATION1
12RELION1.4RECONSTRUCTION1
13PHENIXMODEL REFINEMENT1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 6600
SymmetryPoint symmetry: I
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 3582 / Algorithm: BACK PROJECTION / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingOverall b value: 174 / Ref protocol: RIGID BODY FIT / Ref 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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