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- EMDB-6423: The U4 antibody epitope on human papillomavirus 16 identified by ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6423
TitleThe U4 antibody epitope on human papillomavirus 16 identified by cryo-EM
Map dataQuasi-HPV16
Sample
  • Sample: Quasi-HPV16
  • Virus: Human papillomavirus type 16
KeywordsHPV16 / Cryo-EM / antibody / U4 / neutralization / Fab
Biological speciesHuman papillomavirus type 16
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsGuan J / Bywaters SM / Brendle SA / Lee H / Ashley RE / Christensen ND / Hafenstein S
CitationJournal: J Virol / Year: 2015
Title: The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy.
Authors: Jian Guan / Stephanie M Bywaters / Sarah A Brendle / Hyunwook Lee / Robert E Ashley / Neil D Christensen / Susan Hafenstein /
Abstract: The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16. ...The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.
IMPORTANCE: Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the ...IMPORTANCE: Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.
History
DepositionAug 11, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseOct 7, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 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

400_6423.gif

Downloads & links

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Map

FileDownload / File: emd_6423.map.gz / Format: CCP4 / Size: 412 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationQuasi-HPV16
Voxel sizeX=Y=Z: 1.48 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-2.01456308 - 4.23273659
Average (Standard dev.)0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-240-240-240
Dimensions480480480
Spacing480480480
CellA=B=C: 710.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.481.481.48
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z710.400710.400710.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-240-240-240
NC/NR/NS480480480
D min/max/mean-2.0154.2330.000

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Supplemental data

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Sample components

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Entire : Quasi-HPV16

EntireName: Quasi-HPV16
Components
  • Sample: Quasi-HPV16
  • Virus: Human papillomavirus type 16

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Supramolecule #1000: Quasi-HPV16

SupramoleculeName: Quasi-HPV16 / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 29.8 MDa

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Supramolecule #1: Human papillomavirus type 16

SupramoleculeName: Human papillomavirus type 16 / type: virus / ID: 1 / NCBI-ID: 333760 / Sci species name: Human papillomavirus type 16 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Sci species serotype: HPV16
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Homo sapiens (human) / Recombinant strain: HEK293TT / Recombinant plasmid: SV40
Molecular weightTheoretical: 29.8 MDa
Virus shellShell ID: 1 / Name: L1 L2 / Diameter: 590 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: 1 M NaCl, 200 mM Tris
GridDetails: glow-discharged holey carbon support grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 102 K / Instrument: GATAN CRYOPLUNGE 3

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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: 3.05 µm / Nominal defocus min: 0.84 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
DateSep 4, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 297 / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: auto3dem, RELION, EMAN2
Details: Semi-automatic particle selection was performed using e2boxer.py, followed by particle boxing, linearization, normalization, and apodization of the images using Robem. Defocus and ...Details: Semi-automatic particle selection was performed using e2boxer.py, followed by particle boxing, linearization, normalization, and apodization of the images using Robem. Defocus and astigmatism values for contrast transfer function (CTF) correction were assessed for the extracted particles using ctffind3. The icosahedrally-averaged reconstruction was initiated using a random model generated with setup_rmc and reached 13 A resolution (FSC 0.5, RELION).
Number images used: 1895
DetailsThe particles were selected using semi-automatic program e2boxer.py (EMAN2).

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