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- EMDB-5624: Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4... -

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Entry
Database: EMDB / ID: EMD-5624
TitleBroadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans
Map dataReconstruction of SOSIP.664 HIV-1 envelope trimer in complex with broadly neutralizing PGT122 Fab
Sample
  • Sample: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
  • Protein or peptide: BG505 HIV-1 Env SOSIP.664
KeywordsSingle particle analysis / uranyl formate / tilt series / antibody / HIV gp120 trimer / Fab
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / negative staining / Resolution: 14.0 Å
AuthorsKhayat R / Lee JH / Julien JP / Wilson IA / Ward AB
CitationJournal: PLoS Pathog / Year: 2013
Title: Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.
Authors: Jean-Philippe Julien / Devin Sok / Reza Khayat / Jeong Hyun Lee / Katie J Doores / Laura M Walker / Alejandra Ramos / Devan C Diwanji / Robert Pejchal / Albert Cupo / Umesh Katpally / Rafael ...Authors: Jean-Philippe Julien / Devin Sok / Reza Khayat / Jeong Hyun Lee / Katie J Doores / Laura M Walker / Alejandra Ramos / Devan C Diwanji / Robert Pejchal / Albert Cupo / Umesh Katpally / Rafael S Depetris / Robyn L Stanfield / Ryan McBride / Andre J Marozsan / James C Paulson / Rogier W Sanders / John P Moore / Dennis R Burton / Pascal Poignard / Andrew B Ward / Ian A Wilson /
Abstract: New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, ...New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.
History
DepositionMar 27, 2013-
Header (metadata) releaseApr 10, 2013-
Map releaseJul 3, 2013-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.87
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.87
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5624.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of SOSIP.664 HIV-1 envelope trimer in complex with broadly neutralizing PGT122 Fab
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 3.87 / Movie #1: 3.87
Minimum - Maximum-1.24171531 - 12.395413400000001
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-1.24212.3950.000

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Supplemental data

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Sample components

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Entire : Fab fragment of broadly neutralizing antibody PGT122 in complex w...

EntireName: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
Components
  • Sample: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
  • Protein or peptide: BG505 HIV-1 Env SOSIP.664

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Supramolecule #1000: Fab fragment of broadly neutralizing antibody PGT122 in complex w...

SupramoleculeName: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: one SOSIP.664 trimer binds 3 PGT122 Fabs / Number unique components: 2
Molecular weightExperimental: 350 KDa / Theoretical: 350 KDa / Method: SDS-PAGE

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Macromolecule #1: BG505 HIV-1 Env SOSIP.664

MacromoleculeName: BG505 HIV-1 Env SOSIP.664 / type: protein_or_peptide / ID: 1 / Details: Bound to Fab portion of PGT122 antibody / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: BG505 / synonym: HIV-1
Molecular weightExperimental: 220 KDa / Theoretical: 220 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293S GnT I-deficient cells

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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

Concentration0.1 mg/mL
BufferpH: 7 / Details: 20 mM Tris-HCl, pH 7.0, 50 mM NaCl
StainingType: NEGATIVE
Details: Grids were briefly adsorbed with protein, wicked, and stained with 2% Nano-W for 30 seconds.
GridDetails: 400 Cu mesh grid with thin carob support, glow discharged in natural atmosphere.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.72 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
TemperatureMin: 293 K / Max: 294 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Astigmatism of objective lens was corrected at 100,000x
Legacy - Electron beam tilt params: -2
DateApr 30, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 0.109 µm / Number real images: 340 / Average electron dose: 16 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final two d classificationNumber classes: 64
Final angle assignmentDetails: SPIDER: theta 45 degrees, phi 45 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx
Details: Final map was calculated from a single data set. Multiple data sets produced indistinguishable maps, but data were not combined.
Number images used: 10413
DetailsAll particles were automatically selected from micrographs with DoG Picker [63]. Contrast Transfer function (CTF) estimation for the untilted and tilted micrographs was determined with ctffind3 and ctftilt [64]. Particles were binned by 4 (80x80 sized boxes) and reference-free 2D class averages were calculated using the Sparx package (Fig. S4) [65]. Forty ab initio models were generated from the final reference-free 2D class averages using the EMAN2 package. Each model was then refined against the reference-free 2D class averages using Sparx [65,66]. The model exhibiting Fab-like density was used as the initial model for iterative image reconstruction against the CTF-corrected particles using Sparx [65].

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