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- EMDB-5920: Negative stain reconstruction of PGT151 Fab in complex with cleav... -

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Basic information

Entry
Database: EMDB / ID: EMD-5920
TitleNegative stain reconstruction of PGT151 Fab in complex with cleaved IAVI C22 Env trimer containing the transmembrane region
Map dataNegative stain reconstruction of IAVI C22 dCT Env trimer with PGT151 Fab bound
Sample
  • Sample: Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved IAVI C22 Env trimer with transmembrane region
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: PGT151 Antibody Fab fragment
KeywordsHIV-1 envelope glycoprotein trimer / broadly neutralizing antibody / PGT151
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsLee JH / Blattner C / Wilson IA / Ward AB
CitationJournal: Immunity / Year: 2014
Title: Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.
Authors: Claudia Blattner / Jeong Hyun Lee / Kwinten Sliepen / Ronald Derking / Emilia Falkowska / Alba Torrents de la Peña / Albert Cupo / Jean-Philippe Julien / Marit van Gils / Peter S Lee / ...Authors: Claudia Blattner / Jeong Hyun Lee / Kwinten Sliepen / Ronald Derking / Emilia Falkowska / Alba Torrents de la Peña / Albert Cupo / Jean-Philippe Julien / Marit van Gils / Peter S Lee / Wenjie Peng / James C Paulson / Pascal Poignard / Dennis R Burton / John P Moore / Rogier W Sanders / Ian A Wilson / Andrew B Ward /
Abstract: All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a ...All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.
History
DepositionMar 10, 2014-
Header (metadata) releaseApr 16, 2014-
Map releaseMay 7, 2014-
UpdateMay 28, 2014-
Current statusMay 28, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.27
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.27
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5920.gif

Downloads & links

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Map

FileDownload / File: emd_5920.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of IAVI C22 dCT Env trimer with PGT151 Fab bound
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 80 pix.
= 328. Å		4.1 Å/pix.
x 80 pix.
= 328. Å		4.1 Å/pix.
x 80 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.27 / Movie #1: 2.27
Minimum - Maximum-2.16076517 - 11.652725220000001
Average (Standard dev.)0.00000001 (±0.7889486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-10-10-10
Dimensions808080
Spacing808080
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-10-10-10
NC/NR/NS808080
D min/max/mean-2.16111.6530.000

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Supplemental data

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Sample components

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Entire : Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved...

EntireName: Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved IAVI C22 Env trimer with transmembrane region
Components
  • Sample: Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved IAVI C22 Env trimer with transmembrane region
  • Protein or peptide: HIV-1 Envelope glycoprotein
  • Protein or peptide: PGT151 Antibody Fab fragment

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Supramolecule #1000: Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved...

SupramoleculeName: Fab fragment of HIV-1 Env antibody PGT151 in complex with cleaved IAVI C22 Env trimer with transmembrane region
type: sample / ID: 1000 / Oligomeric state: 2 Fabs bind one Env Trimer / Number unique components: 2
Molecular weightTheoretical: 520 KDa

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Macromolecule #1: HIV-1 Envelope glycoprotein

MacromoleculeName: HIV-1 Envelope glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: Env
Details: The trimer was co-expressed with Furin and Tat. Cleaved trimers were isolated using PGT151 antibody. Contains the transmembrane domain, but not the cytoplasmic domain.
Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: IAVI C22 / synonym: HIV-1
Molecular weightTheoretical: 420 MDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F / Recombinant plasmid: pSVIIIenv

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Macromolecule #2: PGT151 Antibody Fab fragment

MacromoleculeName: PGT151 Antibody Fab fragment / type: protein_or_peptide / ID: 2 / Name.synonym: PGT151 Fab / Details: Heavy chain-light chain dimer of PGT151 Fab / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: B-cells
Molecular weightTheoretical: 49.5 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, 150 mM NaCl, 0.1% DDM
StainingType: NEGATIVE / Details: Sample diluted to <0.001% DDM, immediately applied to grid briefly, then stained for 30-45 seconds with 2% uranyl formate.
GridDetails: plasma-cleaned carbon coated 400 mesh grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureMin: 293 K / Max: 293 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism corrected at 52,000 times magnification
DetailsData collected in 10 degree tilt increments
DateOct 19, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 0.205 µm / Number real images: 1114 / Average electron dose: 26 e/Å2
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: 1.0 µm / Nominal defocus min: 0.55 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 40

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Image processing

CTF correctionDetails: not corrected
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: XMIPP, EMAN, EMAN2, Sparx
Details: Reference free 2D class averages generated using XMIPP and Sparx to sort particles. EMAN2 was used to generate an initial model followed by projection matching carried out in EMAN.
Number images used: 6511

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Atomic model buiding 1

Initial modelPDB ID:

3j5m


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: F / Chain - #4 - Chain ID: I / Chain - #5 - Chain ID: J
SoftwareName: Chimera
DetailsThe PGV04 Fabs in the structure were removed prior to fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4nug


Chain - #0 - Chain ID: H / Chain - #1 - Chain ID: L
SoftwareName: Chimera
DetailsTwo Fabs were fit independently of each other into the EM reconstruction.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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