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- EMDB-6500: Negative stain EM reconstruction of HIV-1 Env AMC008 SOSIP.v4 in ... -

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Entry
Database: EMDB / ID: EMD-6500
TitleNegative stain EM reconstruction of HIV-1 Env AMC008 SOSIP.v4 in complex with Fabs 35O22 and PGV04
Map dataReconstruction of HIV-1 Env AMC008 SOSIP.v4 in complex with broadly-neutralzing antibody Fabs 35O22 and PGV04
Sample
  • Sample: HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing antibody Fabs 35O22 and PGV04
  • Protein or peptide: HIV-1 AMC008 Env SOSIP.v4
  • Protein or peptide: Anti-HIV-1 antibody PGV04 Fab
  • Protein or peptide: Anti-HIV-1 antibody 35O22 Fab
KeywordsHIV-1 / Env / SOSIP / broadly-neutralizing antibody / trimer
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 15.0 Å
Authorsde Taeye SW / Ozorowski G / de la Pena AT / Guttman M / Julien J-P / van den Kerkhof TLGM / Burger J / Pritchard LK / Pugach P / Yasmeen A ...de Taeye SW / Ozorowski G / de la Pena AT / Guttman M / Julien J-P / van den Kerkhof TLGM / Burger J / Pritchard LK / Pugach P / Yasmeen A / Bontjer I / Torres JL / Arendt H / DeStefano J / Koff WC / Schuitemaker H / Eggink D / Berkhout B / Dean H / LaBranche C / Crispin M / Montefiori DC / Klasse PJ / Lee KK / Moore JP / Wilson IA / Ward AB / Sanders RW
CitationJournal: Cell / Year: 2015
Title: Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.
Authors: Steven W de Taeye / Gabriel Ozorowski / Alba Torrents de la Peña / Miklos Guttman / Jean-Philippe Julien / Tom L G M van den Kerkhof / Judith A Burger / Laura K Pritchard / Pavel Pugach / ...Authors: Steven W de Taeye / Gabriel Ozorowski / Alba Torrents de la Peña / Miklos Guttman / Jean-Philippe Julien / Tom L G M van den Kerkhof / Judith A Burger / Laura K Pritchard / Pavel Pugach / Anila Yasmeen / Jordan Crampton / Joyce Hu / Ilja Bontjer / Jonathan L Torres / Heather Arendt / Joanne DeStefano / Wayne C Koff / Hanneke Schuitemaker / Dirk Eggink / Ben Berkhout / Hansi Dean / Celia LaBranche / Shane Crotty / Max Crispin / David C Montefiori / P J Klasse / Kelly K Lee / John P Moore / Ian A Wilson / Andrew B Ward / Rogier W Sanders /
Abstract: The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. ...The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.
History
DepositionNov 2, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseJun 22, 2016-
UpdateJun 22, 2016-
Current statusJun 22, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 12.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 12.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6500.tif

Downloads & links

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Map

FileDownload / File: emd_6500.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of HIV-1 Env AMC008 SOSIP.v4 in complex with broadly-neutralzing antibody Fabs 35O22 and PGV04
Voxel sizeX=Y=Z: 1.57 Å
Density
Contour LevelBy AUTHOR: 12.4 / Movie #1: 12.5
Minimum - Maximum-32.386253359999998 - 78.942092900000006
Average (Standard dev.)1.22258127 (±8.015358920000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 251.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.571.571.57
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z251.200251.200251.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-32.38678.9421.223

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Supplemental data

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Sample components

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Entire : HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing an...

EntireName: HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing antibody Fabs 35O22 and PGV04
Components
  • Sample: HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing antibody Fabs 35O22 and PGV04
  • Protein or peptide: HIV-1 AMC008 Env SOSIP.v4
  • Protein or peptide: Anti-HIV-1 antibody PGV04 Fab
  • Protein or peptide: Anti-HIV-1 antibody 35O22 Fab

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Supramolecule #1000: HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing an...

SupramoleculeName: HIV-1 Env AMC008 SOSIP.v4 in complex with broadly neutralizing antibody Fabs 35O22 and PGV04
type: sample / ID: 1000
Details: All components were purified by size-exclusion chromatography prior to complex formation.
Oligomeric state: 3 of each Fab bound to a trimer of HIV-1 Env SOSIP.v4
Number unique components: 3
Molecular weightTheoretical: 720 KDa
Method: Estimation based on average molecular weight of Fab and average mass of SOSIP trimer including glycan mass

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Macromolecule #1: HIV-1 AMC008 Env SOSIP.v4

MacromoleculeName: HIV-1 AMC008 Env SOSIP.v4 / type: protein_or_peptide / ID: 1 / Name.synonym: HIV-1 gp140 / Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: AMC008
Molecular weightTheoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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Macromolecule #2: Anti-HIV-1 antibody PGV04 Fab

MacromoleculeName: Anti-HIV-1 antibody PGV04 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Oligomeric state: Heterodimer of light and heavy 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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Macromolecule #3: Anti-HIV-1 antibody 35O22 Fab

MacromoleculeName: Anti-HIV-1 antibody 35O22 Fab / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Oligomeric state: Heterodimer of light and heavy 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

Concentration0.03 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE
Details: Grids were stained with 2% w/v uranyl formate for 60 seconds.
GridDetails: 400 mesh copper grid with thin carbon support, glow-discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeOTHER
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -50
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 92,000 times magnification.
DetailsTilt series of -50, -40, -30, -20, -10, 0 degrees
DateFeb 19, 2015
Image recordingCategory: CCD / Film or detector model: FEI CETA (4k x 4k) / Number real images: 152 / Average electron dose: 25 e/Å2
Tilt angle max0

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 24522
DetailsParticles were selected using an automatic selection program and aligned into class averages using Iterative MSA/MRA. Classes containing fully decorated (3 molecules of each Fab per trimer) complexes were used to generate a common lines model that was later refined using particles from those classes.

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