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- EMDB-5993: Electron cryo-microscopy of human papillomavirus 16 and H16.V5 Fa... -

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Basic information

Entry
Database: EMDB / ID: EMD-5993
TitleElectron cryo-microscopy of human papillomavirus 16 and H16.V5 Fab fragments
Map dataReconstruction of mature human papillomavirus 16 capsid
Sample
  • Sample: Mature HPV16 quasivirus capsid
  • Virus: Human papillomavirus 16
Keywordsvirus-Fab complex / neutralization antibody / maturation
Biological speciesHuman papillomavirus 16
Methodsingle particle reconstruction / cryo EM / Resolution: 12.6 Å
AuthorsLee H / Brendle SA / Bywaters SM / Guan J / Ashley RE / Yoder JD / Makhov AM / Conway JF / Christensen ND / Hafenstein S
CitationJournal: J Virol / Year: 2015
Title: A cryo-electron microscopy study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment.
Authors: Hyunwook Lee / Sarah A Brendle / Stephanie M Bywaters / Jian Guan / Robert E Ashley / Joshua D Yoder / Alexander M Makhov / James F Conway / Neil D Christensen / Susan Hafenstein /
Abstract: Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV ...Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites.
IMPORTANCE: Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization ...IMPORTANCE: Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5.
History
DepositionJun 20, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseNov 26, 2014-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_5993.map.gz / Format: CCP4 / Size: 502.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mature human papillomavirus 16 capsid
Voxel sizeX=Y=Z: 1.48 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-4.7340517 - 6.09024334
Average (Standard dev.)0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions513513513
Spacing513513513
CellA=B=C: 759.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.481.481.48
M x/y/z513513513
origin x/y/z0.0000.0000.000
length x/y/z759.240759.240759.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS513513513
D min/max/mean-4.7346.0900.000

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Supplemental data

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Sample components

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Entire : Mature HPV16 quasivirus capsid

EntireName: Mature HPV16 quasivirus capsid
Components
  • Sample: Mature HPV16 quasivirus capsid
  • Virus: Human papillomavirus 16

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Supramolecule #1000: Mature HPV16 quasivirus capsid

SupramoleculeName: Mature HPV16 quasivirus capsid / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 27 MDa

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Supramolecule #1: Human papillomavirus 16

SupramoleculeName: Human papillomavirus 16 / type: virus / ID: 1 / NCBI-ID: 337041 / Sci species name: Human papillomavirus 16 / Database: NCBI / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 27 MDa
Virus shellShell ID: 1 / Diameter: 600 Å / T number (triangulation number): 7

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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

Concentration1.0 mg/mL
BufferpH: 7.4
Details: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4
GridDetails: glow-discharged holey carbon Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 102 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 0.7 seconds before plunging.

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.75 µm / Nominal defocus min: 0.57 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
DateOct 10, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 170 / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.6 Å / Resolution method: OTHER / Software - Name: Auto3dem
Details: Semi-automatic particle selection was performed using e2boxer.py to obtain the particle coordinates, followed by particle boxing, linearization, normalization, and apodization of the images ...Details: Semi-automatic particle selection was performed using e2boxer.py to obtain the particle coordinates, followed by particle boxing, linearization, normalization, and apodization of the images using Robem. Defocus and astigmatism values to perform contrast transfer function (CTF) correction were assessed using Robem for the extracted particles. The icosahedrally averaged reconstructions were initiated using a random model generated with setup_rmc and reached 13 A resolution estimated at a Fourier Shell Correlation (FSC) of 0.5. For the last step of refinement, the final maps were CTF-corrected using a B factor of 200 A2.
Number images used: 4737
DetailsThe particles were selected using semi-automatic program e2boxer.py (EMAN2).

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