National Institutes of Health/Office of the Director
United States
Citation
Journal: 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.
#200 - Aug 2016 Quasisymmetry in Icosahedral Viruses similarity (2)
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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
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
Idetical with deposited unit in distinct coordinate
icosahedral asymmetric unit
Type
Name
Symmetry operation
Number
point symmetry operation
1
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 mass
Number of molelcules
Total (without water)
1,682,959
30
Polymers
1,682,959
30
Non-polymers
0
0
Water
0
Type
Name
Symmetry operation
Number
point symmetry operation
5
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 mass
Number of molelcules
Total (without water)
2,019,550
36
Polymers
2,019,550
36
Non-polymers
0
0
Water
0
Type
Name
Symmetry operation
Number
point symmetry operation
6
5
Idetical with deposited unit in distinct coordinate
icosahedral asymmetric unit, std point frame
Type
Name
Symmetry operation
Number
transform to point frame
1
Symmetry
Point symmetry: (Schoenflies symbol: I (icosahedral))
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Components
#1: Protein
MajorcapsidproteinL1
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
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: 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 virus
Empty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Buffer solution
pH: 7.4
Buffer component
Conc.: 1 X / Name: PBS
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Instrument: 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
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm
Specimen holder
Cryogen: NITROGEN
Image recording
Electron 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
ID
Name
Version
Category
Details
1
cryoSPARC
2.8.3
particleselection
Templatepickerfrommanualpicks
4
cryoSPARC
2.8.3
CTFcorrection
CTFFIND4
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 202705
3D reconstruction
Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181299 / Symmetry type: POINT
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