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- EMDB-6118: Electron cryo-microscopy of human papillomavirus 16 and H16.V5 Fa... -

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Basic information

Entry
Database: EMDB / ID: EMD-6118
TitleElectron cryo-microscopy of human papillomavirus 16 and H16.V5 Fab fragments
Map dataReconstruction of mature human papillomavirus 16 capsid complexed with Fab fragments after 72 hr incubation
Sample
  • Sample: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs
  • Virus: Human papillomavirus 16
  • Protein or peptide: H16.V5 Fab
Keywordsvirus-Fab complex / neutralization antibody / maturation
Biological speciesMus musculus (house mouse) / Human papillomavirus 16
Methodsingle particle reconstruction / cryo EM / Resolution: 15.8 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
DepositionSep 30, 2014-
Header (metadata) releaseOct 8, 2014-
Map releaseDec 3, 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

400_6118.gif

Downloads & links

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Map

FileDownload / File: emd_6118.map.gz / Format: CCP4 / Size: 112.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mature human papillomavirus 16 capsid complexed with Fab fragments after 72 hr incubation
Voxel sizeX=Y=Z: 2.86 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-5.65556288 - 5.93166399
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-155-155-155
Dimensions311311311
Spacing311311311
CellA=B=C: 889.45996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.862.862.86
M x/y/z311311311
origin x/y/z0.0000.0000.000
length x/y/z889.460889.460889.460
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-155-155-155
NC/NR/NS311311311
D min/max/mean-5.6565.9320.000

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Supplemental data

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Sample components

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Entire : Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs

EntireName: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs
Components
  • Sample: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs
  • Virus: Human papillomavirus 16
  • Protein or peptide: H16.V5 Fab

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Supramolecule #1000: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs

SupramoleculeName: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs
type: sample / ID: 1000
Oligomeric state: Three hundred H16.V5 Fabs bind to one HPV16 capsid
Number unique components: 2
Molecular weightTheoretical: 42 MDa

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Supramolecule #1: Human papillomavirus 16

SupramoleculeName: Human papillomavirus 16 / type: virus / ID: 1
Details: The virus sample was incubated with excessive Fab fragments for 72 hrs.
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 / Name: L1/L2 / Diameter: 600 Å / T number (triangulation number): 7

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Macromolecule #1: H16.V5 Fab

MacromoleculeName: H16.V5 Fab / type: protein_or_peptide / ID: 1 / Number of copies: 300 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / Cell: hybridoma
Molecular weightTheoretical: 50 KDa

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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: 5.18 µm / Nominal defocus min: 1.63 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
DateAug 18, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 102 / Average electron dose: 15 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.8 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 14 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: 2075
DetailsThe particles were selected using semi-automatic program e2boxer.py (EMAN2).

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