|Entry||Database: EMDB / ID: 6119|
|Title||Electron cryo-microscopy of human papillomavirus 16 and H16.V5 Fab fragments|
|Map data||Reconstruction of mature human papillomavirus 16 capsid complexed with Fab fragments after 1 hr incubation|
|Sample||Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs:|
virus / H16.V5 Fab
|Keywords||virus-Fab complex / neutralization antibody / maturation|
|Source||Human papillomavirus 16 / Mus musculus (house mouse)|
|Method||single particle reconstruction / cryo EM / 14.7 Å resolution|
|Authors||Lee H / Brendle SA / Bywaters SM / Guan J / Ashley RE / Yoder JD / Makhov AM / Conway JF / Christensen ND / Hafenstein S|
|Citation||Journal: 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 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.
|Date||Deposition: Sep 30, 2014 / Header (metadata) release: Oct 8, 2014 / Map release: Dec 3, 2014 / Last update: May 27, 2015|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6119.map.gz (map file in CCP4 format, 117502 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.86 Å|
CCP4 map header:
-Entire Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs
|Entire||Name: Mature HPV16 quasivirus capsid complexed with H16.V5 Fabs|
Number of components: 2
Oligomeric State: Three hundred H16.V5 Fabs bind to one HPV16 capsid
|Mass||Theoretical: 42 MDa|
-Component #1: virus, Human papillomavirus 16
|Virus||Name: Human papillomavirus 16Papillomaviridae / Class: VIRION|
Details: The virus sample was incubated with excessive Fab fragments for 1 hr.
Enveloped: No / Empty: No / Isolate: OTHER
|Mass||Theoretical: 27 MDa|
|Species||Species: Human papillomavirus 16|
|Source (natural)||Host Species: Homo sapiens (human) / Host category: VERTEBRATES|
|Shell #1||Name of element: L1/L2 / Diameter: 600 Å / T number(triangulation number): 7|
-Component #2: protein, H16.V5 Fab
|Protein||Name: H16.V5 Fab / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 300|
|Mass||Theoretical: 50 kDa|
|Source||Species: Mus musculus (house mouse)|
|Source (natural)||Cell: hybridoma|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 1 mg/ml|
Buffer solution: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4
|Support film||glow-discharged holey carbon Quantifoil grids|
|Vitrification||Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 102 K / Humidity: 90 % / Method: Blot for 0.7 seconds before plunging.|
-Electron microscopy imaging
|Imaging||Microscope: JEOL 2100 / Date: Aug 13, 2014|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 200 kV / Electron dose: 15 e/Å2 / Illumination mode: SPOT SCAN|
|Lens||Magnification: 40000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1190 - 5670 nm|
|Specimen Holder||Model: GATAN LIQUID NITROGEN / Temperature: 95 K|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Image acquisition||Number of digital images: 138|
|Processing||Method: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2075|
Details: The particles were selected using semi-automatic program e2boxer.py (EMAN2).
|3D reconstruction||Algorithm: Cross-common lines / Software: Auto3dem / CTF correction: Each particle|
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.
Resolution: 14.7 Å / Resolution method: FSC 0.5, semi-independent
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