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- EMDB-2241: Negative stain reconstruction of PG9 Fab in complex with HIV-1 SO... -

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Basic information

Entry
Database: EMDB / ID: EMD-2241
TitleNegative stain reconstruction of PG9 Fab in complex with HIV-1 SOSIP gp140 trimer
Map dataReconstruction of SOSIP gp140 trimer in complex with PG9 Fab
Sample
  • Sample: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
  • Protein or peptide: Broadly Neutralizing Antibody PG9
  • Protein or peptide: HIV-1 SOSIP.664
KeywordsHuman Immunodeficiency Virus / HIV / Broadly neutralizing antibody / PG9 / Env
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / identical protein binding / membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHomo sapiens (human) / Human immunodeficiency virus 1
Methodsingle particle reconstruction / negative staining / Resolution: 18.1 Å
AuthorsLee JH / Julien JP / Cupo A / Moore JP / Wilson IA / Ward AB
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9.
Authors: Jean-Philippe Julien / Jeong Hyun Lee / Albert Cupo / Charles D Murin / Ronald Derking / Simon Hoffenberg / Michael J Caulfield / C Richter King / Andre J Marozsan / Per Johan Klasse / ...Authors: Jean-Philippe Julien / Jeong Hyun Lee / Albert Cupo / Charles D Murin / Ronald Derking / Simon Hoffenberg / Michael J Caulfield / C Richter King / Andre J Marozsan / Per Johan Klasse / Rogier W Sanders / John P Moore / Ian A Wilson / Andrew B Ward /
Abstract: PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. ...PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody-trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region.
History
DepositionDec 7, 2012-
Header (metadata) releaseJan 9, 2013-
Map releaseMar 6, 2013-
UpdateMar 20, 2013-
Current statusMar 20, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2241.png

Downloads & links

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Map

FileDownload / File: emd_2241.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of SOSIP gp140 trimer in complex with PG9 Fab
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.18 Å/pix.
x 160 pix.
= 348.8 Å		2.18 Å/pix.
x 160 pix.
= 348.8 Å		2.18 Å/pix.
x 160 pix.
= 348.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.18 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.03 / Movie #1: 1.03
Minimum - Maximum-1.92045236 - 10.45579624
Average (Standard dev.)0.0 (±0.53768271)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-37-37-37
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-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-37-37-37
NC/NR/NS160160160
D min/max/mean-1.92010.4560.000

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Supplemental data

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Sample components

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Entire : Fab fragment of broadly neutralizing antibody PG9 bound to BG505 ...

EntireName: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
Components
  • Sample: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
  • Protein or peptide: Broadly Neutralizing Antibody PG9
  • Protein or peptide: HIV-1 SOSIP.664

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Supramolecule #1000: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 ...

SupramoleculeName: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
type: sample / ID: 1000 / Oligomeric state: One Fab binds one SOSIP trimer / Number unique components: 2
Molecular weightExperimental: 387 KDa / Theoretical: 392 KDa
Method: Size exclusion chromatography with in-line multi-angle light scattering (SEC-MALS)

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Macromolecule #1: Broadly Neutralizing Antibody PG9

MacromoleculeName: Broadly Neutralizing Antibody PG9 / type: protein_or_peptide / ID: 1 / Name.synonym: bnAb PG9
Details: Recombinantly expressed antibody Fab fragment. Co-transfected with gene encoding tyroslprotein sulfotransferase 1 (TPST1)
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: B-Cell
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: HEK293T (human)

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Macromolecule #2: HIV-1 SOSIP.664

MacromoleculeName: HIV-1 SOSIP.664 / type: protein_or_peptide / ID: 2 / Name.synonym: gp140 / Details: Cotransfected with pcDNA3.1 Furin. / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: BG505 / synonym: HIV-1
Molecular weightExperimental: 120 KDa / Theoretical: 140 KDa
Recombinant expressionOrganism: HEK293S GnTI -/- / Recombinant plasmid: pP14

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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: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE / Details: 30 sec Nano-W
GridDetails: 400 Cu mesh grid with carbon support, glow discharged at 20 mA for 30 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 293 K / Max: 293 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 100,000x
DetailsImages taken from 0 to -55 degrees in 5 degree tilt increments.
DateJul 31, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 0.109 µm / Number real images: 1283 / Average electron dose: 30 e/Å2 / Details: Images recorded on CCD / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were picked using DogPicker then processed with Xmipp and EMAN
CTF correctionDetails: Not corrected
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp, EMAN / Number images used: 33431

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