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- EMDB-5983: Structure of 2G12 (Fab)2 in Complex with Soluble and Fully Glycos... -

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Entry
Database: EMDB / ID: EMD-5983
TitleStructure of 2G12 (Fab)2 in Complex with Soluble and Fully Glycosylated HIV-1 Env by Negative-Stain Single Particle Electron Microscopy
Map dataReconstruction of HIV-1 Env SOSIP BG505.664 bound to soluble, two-domain CD4 (domains 1 and 2, sCD4) and 2G12 Fabs
Sample
  • Sample: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
  • Protein or peptide: HIV-1 Env
  • Protein or peptide: soluble CD4
  • Protein or peptide: Human Monoclonal Antibody 2G12 IgG1 Fab Fragment
KeywordsHIV-1 / 2G12 / monoclonal antibodies / Envelope / CD4-bound Env
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsMurin CD / Julien JP / Sok D / Stanfield R / Khayat R / Cupo A / Moore JP / Burton DR / Wilson IA / Ward AB
CitationJournal: J Virol / Year: 2014
Title: Structure of 2G12 Fab2 in complex with soluble and fully glycosylated HIV-1 Env by negative-stain single-particle electron microscopy.
Authors: Charles D Murin / Jean-Philippe Julien / Devin Sok / Robyn L Stanfield / Reza Khayat / Albert Cupo / John P Moore / Dennis R Burton / Ian A Wilson / Andrew B Ward /
Abstract: The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody ...The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Å resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Å of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope.
IMPORTANCE: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is ...IMPORTANCE: HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.
History
DepositionJun 3, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseAug 6, 2014-
UpdateSep 9, 2015-
Current statusSep 9, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5983.tif

Downloads & links

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Map

FileDownload / File: emd_5983.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of HIV-1 Env SOSIP BG505.664 bound to soluble, two-domain CD4 (domains 1 and 2, sCD4) and 2G12 Fabs
Voxel sizeX=Y=Z: 4.35 Å
Density
Contour LevelBy AUTHOR: 4.1 / Movie #1: 4.1
Minimum - Maximum-3.92196393 - 13.6949892
Average (Standard dev.)0.00000001 (±0.9999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 417.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.354.354.35
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z417.600417.600417.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-3.92213.6950.000

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Supplemental data

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Sample components

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Entire : Fab fragment of 2G12 monoclonal antibody and soluble, two-domain ...

EntireName: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
Components
  • Sample: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
  • Protein or peptide: HIV-1 Env
  • Protein or peptide: soluble CD4
  • Protein or peptide: Human Monoclonal Antibody 2G12 IgG1 Fab Fragment

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Supramolecule #1000: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain ...

SupramoleculeName: Fab fragment of 2G12 monoclonal antibody and soluble, two-domain CD4 (domains 1 and 2, sCD4) bound to HIV-1 Env BG505.664
type: sample / ID: 1000
Oligomeric state: Env trimer bound to three 2G12 domain-swapped Fabs and three sCD4s
Number unique components: 3
Molecular weightExperimental: 730 KDa / Theoretical: 729 KDa / Method: SEC-MALS

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Macromolecule #1: HIV-1 Env

MacromoleculeName: HIV-1 Env / type: protein_or_peptide / ID: 1 / Name.synonym: SOSIP BG505.664 / Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / synonym: HIV-1
Molecular weightExperimental: 360 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F

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Macromolecule #2: soluble CD4

MacromoleculeName: soluble CD4 / type: protein_or_peptide / ID: 2 / Name.synonym: sCD4 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 23 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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Macromolecule #3: Human Monoclonal Antibody 2G12 IgG1 Fab Fragment

MacromoleculeName: Human Monoclonal Antibody 2G12 IgG1 Fab Fragment / type: protein_or_peptide / ID: 3 / Name.synonym: 2G12 Fab / Oligomeric state: Dimer of Fabs / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 100 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: phCMV3

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.03 mg/mL
BufferpH: 7.4 / Details: 150 mM NaCl, 10 mM Tris
StainingType: NEGATIVE
Details: Grids with adsorbed protein were floated on 2% w/v uranyl formate for 30 seconds.
GridDetails: 400 mesh copper grid with thin carbon support, Gatan plasma cleaned
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 52000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder model: HOME BUILD
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DateJul 12, 2012
Image recordingCategory: CCD / Film or detector model: OTHER / Number real images: 149 / Average electron dose: 30 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 100
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Details: Final map was low pass filtered to 20 Angstrom. / Number images used: 5372
DetailsThe particles were selected using automatic selection program Leginon and processed using the Appion system.

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Atomic model buiding 1

Initial modelPDB ID:

2b4c

SoftwareName: Chimera
DetailsThree structures of HIV-1 gp120 bound to sCD4 were fit into the HIV-1 Env trimer portion of the map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1op5
PDB Unreleased entry

SoftwareName: Chimera
Details2G12 Fab structures were fit by rigid body fitting using the UCSF Chimera volume fit option, simulating a map at an estimated resolution of 20 Angstrom.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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