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- EMDB-2853: 3BNC117 Fab in complex with BG505 SOSIP.664 Trimer -

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Basic information

Entry
Database: EMDB / ID: EMD-2853
Title3BNC117 Fab in complex with BG505 SOSIP.664 Trimer
Map dataSingle particle reconstruction (20 Angstrom resolution based on FSC 0.5)
Sample
  • Sample: Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env gp140 BG505 SOSIP.664 trimer
  • Protein or peptide: Soluble HIV-1 Envelope glycoprotein
  • Protein or peptide: Fab of 3BNC117 human monoclonal antibody
KeywordsHIV / Fab / Env trimer
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsOzorowski G / Derking R / Sanders RW / Ward AB
CitationJournal: PLoS Pathog / Year: 2015
Title: Comprehensive antigenic map of a cleaved soluble HIV-1 envelope trimer.
Authors: Ronald Derking / Gabriel Ozorowski / Kwinten Sliepen / Anila Yasmeen / Albert Cupo / Jonathan L Torres / Jean-Philippe Julien / Jeong Hyun Lee / Thijs van Montfort / Steven W de Taeye / Mark ...Authors: Ronald Derking / Gabriel Ozorowski / Kwinten Sliepen / Anila Yasmeen / Albert Cupo / Jonathan L Torres / Jean-Philippe Julien / Jeong Hyun Lee / Thijs van Montfort / Steven W de Taeye / Mark Connors / Dennis R Burton / Ian A Wilson / Per-Johan Klasse / Andrew B Ward / John P Moore / Rogier W Sanders /
Abstract: The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have ...The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.
History
DepositionJan 19, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseMar 11, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2853_3BN...

Downloads & links

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Map

FileDownload / File: emd_2853.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction (20 Angstrom resolution based on FSC 0.5)
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 10.0 / Movie #1: 10
Minimum - Maximum-25.31560898 - 74.804893489999998
Average (Standard dev.)0.39408892 (±5.32740307)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-25.31674.8050.394

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Supplemental data

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Sample components

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Entire : Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env...

EntireName: Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env gp140 BG505 SOSIP.664 trimer
Components
  • Sample: Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env gp140 BG505 SOSIP.664 trimer
  • Protein or peptide: Soluble HIV-1 Envelope glycoprotein
  • Protein or peptide: Fab of 3BNC117 human monoclonal antibody

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Supramolecule #1000: Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env...

SupramoleculeName: Fab of 3BNC117 monoclonal antibody bound to recombinant HIV-1 Env gp140 BG505 SOSIP.664 trimer
type: sample / ID: 1000
Details: Size-exclusion chromatography purified gp140 trimers were used
Oligomeric state: One Fab molecule binds to one BG505 SOSIP.664 gp140 monomer
Number unique components: 2
Molecular weightTheoretical: 570 KDa
Method: Theoretical MW of Fab based on primary sequence; MW of trimer estimated using published values

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Macromolecule #1: Soluble HIV-1 Envelope glycoprotein

MacromoleculeName: Soluble HIV-1 Envelope glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: SOSIP
Details: gp140 trimers were expressed recombinantly and purified using 2G12-affinity and size-exclusion chromatography
Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: BG505 / synonym: HIV-1
Molecular weightTheoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F / Recombinant plasmid: pPPI4

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Macromolecule #2: Fab of 3BNC117 human monoclonal antibody

MacromoleculeName: Fab of 3BNC117 human monoclonal antibody / type: protein_or_peptide / ID: 2
Details: Expressed recombinantly as IgG and cleaved into Fab using papain
Number of copies: 1 / Oligomeric state: Dimer composed of heavy and light chain / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl pH 7.4, 150 mM NaCl
StainingType: NEGATIVE
Details: 3 microliters of 2% w/v uranyl formate were added to grids adsorbed with protein for 45 seconds and blotted
GridDetails: 400 Cu mesh grid with carbon support, plasma cleaned
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -50
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism corrected at 52,000x mag
DateSep 29, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 145 / Average electron dose: 25 e/Å2
Tilt angle max0

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Image processing

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 14956
DetailsParticles were selected using an automatic selection program. A substack was created containing particles with 3 Fab molecules per trimer. An initial common-lines model was created using class averages and further refined using projection matching and an unbinned stack of particles.

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