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- PDB-7kzf: High resolution cryo EM analysis of HPV16 identifies minor struct... -

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Basic information

Entry
Database: PDB / ID: 7kzf
TitleHigh resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility
ComponentsMajor capsid protein L1
KeywordsVIRUS / HPV16 / quasivirus / L1 / capsomer / subparticle
Function / homologyDouble-stranded DNA virus, group I, capsid / Major capsid L1 (late) protein, Papillomavirus / Major capsid L1 (late) superfamily, Papillomavirus / T=7 icosahedral viral capsid / endocytosis involved in viral entry into host cell / virion attachment to host cell / host cell nucleus / structural molecule activity / Major capsid protein L1
Function and homology information
Biological speciesHuman papillomavirus type 16
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsHartmann, S.R. / Goetschius, D.J. / Hafenstein, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director United States
CitationJournal: Sci Rep / Year: 2021
Title: High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility.
Authors: Daniel J Goetschius / Samantha R Hartmann / Suriyasri Subramanian / Carol M Bator / Neil D Christensen / Susan L Hafenstein /
Abstract: Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte ...Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 24, 2021Provider: repository / Type: Initial release

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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-23081
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
F: Major capsid protein L1
B: Major capsid protein L1
C: Major capsid protein L1
D: Major capsid protein L1
E: Major capsid protein L1
A: Major capsid protein L1


Theoretical massNumber of molelcules
Total (without water)336,5926
Polymers336,5926
Non-polymers00
Water0
1
F: Major capsid protein L1
B: Major capsid protein L1
C: Major capsid protein L1
D: Major capsid protein L1
E: Major capsid protein L1
A: Major capsid protein L1
x 60


Theoretical massNumber of molelcules
Total (without water)20,195,502360
Polymers20,195,502360
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
F: Major capsid protein L1
B: Major capsid protein L1
C: Major capsid protein L1
D: Major capsid protein L1
E: Major capsid protein L1
A: Major capsid protein L1
x 5


  • icosahedral pentamer
  • 1.68 MDa, 30 polymers
Theoretical massNumber of molelcules
Total (without water)1,682,95930
Polymers1,682,95930
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
F: Major capsid protein L1
B: Major capsid protein L1
C: Major capsid protein L1
D: Major capsid protein L1
E: Major capsid protein L1
A: Major capsid protein L1
x 6


  • icosahedral 23 hexamer
  • 2.02 MDa, 36 polymers
Theoretical massNumber of molelcules
Total (without water)2,019,55036
Polymers2,019,55036
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
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Major capsid protein L1


Mass: 56098.617 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human papillomavirus type 16 / Gene: L1 / Cell line (production host): 293TT / Production host: Homo sapiens (human) / References: UniProt: P03101

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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 papillomavirus type 16 / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Human papillomavirus type 16
Source (recombinant)Organism: Homo sapiens (human) / Cell: 293TT
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.4
Buffer componentConc.: 1 X / Name: PBS
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10143

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.8.3particle selectionTemplate picker from manual picks
4cryoSPARC2.8.3CTF correctionCTFFIND4
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 202705
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181299 / Symmetry type: POINT

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