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- PDB-3j7e: Electron cryo-microscopy of human papillomavirus 16 and H16.V5 Fa... -

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Basic information

Entry
Database: PDB / ID: 3j7e
TitleElectron cryo-microscopy of human papillomavirus 16 and H16.V5 Fab fragments
Components
  • H16.V5 Fab heavy chain
  • H16.V5 Fab light chain
KeywordsIMMUNE SYSTEM / HPV16.V5 Fab variable domain / HI and FG loops / HPV16 capsid / virus-Fab complex / neutralization antibody / maturation
Function / homology
Function and homology information


Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
IgM heavy chain variable region
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsLee, H. / Brendle, S.A. / Bywaters, S.M. / Christensen, N.D. / 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 23, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 26, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 3, 2014Group: Database references
Revision 1.2Mar 18, 2015Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Assembly

Deposited unit
L: H16.V5 Fab light chain
H: H16.V5 Fab heavy chain
A: H16.V5 Fab light chain
B: H16.V5 Fab heavy chain
C: H16.V5 Fab light chain
D: H16.V5 Fab heavy chain
E: H16.V5 Fab light chain
F: H16.V5 Fab heavy chain


Theoretical massNumber of molelcules
Total (without water)104,4858
Polymers104,4858
Non-polymers00
Water0
1
L: H16.V5 Fab light chain
H: H16.V5 Fab heavy chain
A: H16.V5 Fab light chain
B: H16.V5 Fab heavy chain
C: H16.V5 Fab light chain
D: H16.V5 Fab heavy chain
E: H16.V5 Fab light chain
F: H16.V5 Fab heavy chain
x 60


Theoretical massNumber of molelcules
Total (without water)6,269,071480
Polymers6,269,071480
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
L: H16.V5 Fab light chain
H: H16.V5 Fab heavy chain
A: H16.V5 Fab light chain
B: H16.V5 Fab heavy chain
C: H16.V5 Fab light chain
D: H16.V5 Fab heavy chain
E: H16.V5 Fab light chain
F: H16.V5 Fab heavy chain
x 5


  • icosahedral pentamer
  • 522 kDa, 40 polymers
Theoretical massNumber of molelcules
Total (without water)522,42340
Polymers522,42340
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
L: H16.V5 Fab light chain
H: H16.V5 Fab heavy chain
A: H16.V5 Fab light chain
B: H16.V5 Fab heavy chain
C: H16.V5 Fab light chain
D: H16.V5 Fab heavy chain
E: H16.V5 Fab light chain
F: H16.V5 Fab heavy chain
x 6


  • icosahedral 23 hexamer
  • 627 kDa, 48 polymers
Theoretical massNumber of molelcules
Total (without water)626,90748
Polymers626,90748
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Antibody
H16.V5 Fab light chain


Mass: 12623.161 Da / Num. of mol.: 4 / Fragment: variable domain / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell: hybridoma
#2: Antibody
H16.V5 Fab heavy chain


Mass: 13497.968 Da / Num. of mol.: 4 / Fragment: variable domain / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell: hybridoma / References: UniProt: K7TH34*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1Mature HPV16 quasivirus capsid complexed with H16.V5 FabsCOMPLEXThree hundred H16.V5 Fabs bind to one HPV16 capsid0
2Human papillomavirus 16PapillomaviridaeVIRUS1
3H16.V5 Fab1
Molecular weightValue: 42 MDa / Experimental value: NO
Details of virusEmpty: NO / Enveloped: NO / Host category: VERTEBRATES / Isolate: OTHER / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionName: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 / pH: 7.4
Details: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow-discharged holey carbon Quantifoil grids
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temp: 102 K / Humidity: 90 %
Details: Blot for 0.7 seconds before plunging into liquid ethane (GATAN CRYOPLUNGE 3).
Method: Blot for 0.7 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: JEOL 2100 / Date: Oct 30, 2013
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 80000 X / Nominal defocus max: 3990 nm / Nominal defocus min: 690 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature: 95 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 411
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Situsmodel fitting
2UCSF Chimeramodel fitting
3Auto3DEM3D reconstruction
4EMAN23D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Cross-common lines / Resolution: 13.6 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 2075 / Nominal pixel size: 1.48 Å / Actual pixel size: 1.48 Å
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. (Single particle--Applied symmetry: I)
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: REFINEMENT PROTOCOL--flexible
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms7356 0 0 0 7356

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