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- EMDB-6252: VRC01 Fab in complex with BG505 SOSIP.664 Trimer -

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Basic information

Entry
Database: EMDB / ID: EMD-6252
TitleVRC01 Fab in complex with BG505 SOSIP.664 Trimer
Map dataVRC01 Fab in complex with BG505 SOSIP.664 Trimer
Sample
  • Sample: Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 BG505 SOSIP.664
  • Protein or peptide: Soluble HIV-1 Envelope glycoprotein
  • Protein or peptide: Fab of VRC01 human monoclonal antibody
KeywordsHIV-1 / Fab / Env trimer
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsTorres JL / Ozorowski 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-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6252.gif

Downloads & links

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Map

FileDownload / File: emd_6252.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVRC01 Fab in complex with BG505 SOSIP.664 Trimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.05 Å/pix.
x 160 pix.
= 328. Å		2.05 Å/pix.
x 160 pix.
= 328. Å		2.05 Å/pix.
x 160 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0 / Movie #1: 6
Minimum - Maximum-9.35602188 - 39.293411249999998
Average (Standard dev.)0.35270497 (±3.06196618)
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-9.35639.2930.353

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Supplemental data

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Sample components

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Entire : Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 B...

EntireName: Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 BG505 SOSIP.664
Components
  • Sample: Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 BG505 SOSIP.664
  • Protein or peptide: Soluble HIV-1 Envelope glycoprotein
  • Protein or peptide: Fab of VRC01 human monoclonal antibody

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Supramolecule #1000: Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 B...

SupramoleculeName: Fab of VRC01 human monoclonal antibody bound to HIV-1 Env gp140 BG505 SOSIP.664
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

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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 / 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 VRC01 human monoclonal antibody

MacromoleculeName: Fab of VRC01 human monoclonal antibody / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Oligomeric state: dimer (heavy chain + 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 uL of protein was applied and blotted; then 2% w/v uranyl formate was applied for 45 seconds.
GridDetails: 400 Cu mesh grid with carbon support, plasma-cleaned
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 52,000x magnification.
DateOct 27, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 174 / Average electron dose: 25 e/Å2
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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

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Image processing

DetailsThe particles were selected using an automatic selection program. Class averages with 3 Fabs bound to one gp140 trimer were placed into a substack and used for 3D reconstruction.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 7861

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