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- EMDB-5462: Cryo-electron tomography of a trimeric soluble HIV Env construct,... -

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Basic information

Entry
Database: EMDB / ID: EMD-5462
TitleCryo-electron tomography of a trimeric soluble HIV Env construct, gp140 SOSIP, in complex with Fab 17b
Map dataReconstruction of soluble KNH1144-SOSIP Env trimers in complex with Fab 17b
Sample
  • Sample: Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.
  • Protein or peptide: Envelope glycoprotein
  • Protein or peptide: Fab portion of monoclonal antibody 17b
KeywordsHIV / gp140 / SOSIP / 17b
Biological speciesHuman immunodeficiency virus 1 / unidentified (others)
Methodsubtomogram averaging / cryo EM / Resolution: 8.8 Å
AuthorsTran EEH / Borgnia MJ / Kuybeda O / Schauder DM / Bartesaghi A / Frank GA / Sapiro G / Milne JLS / Subramaniam S
CitationJournal: PLoS Pathog / Year: 2012
Title: Structural mechanism of trimeric HIV-1 envelope glycoprotein activation.
Authors: Erin E H Tran / Mario J Borgnia / Oleg Kuybeda / David M Schauder / Alberto Bartesaghi / Gabriel A Frank / Guillermo Sapiro / Jacqueline L S Milne / Sriram Subramaniam /
Abstract: HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of ...HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.
History
DepositionAug 6, 2012-
Header (metadata) releaseOct 3, 2012-
Map releaseOct 3, 2012-
UpdateSep 25, 2013-
Current statusSep 25, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 520
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 520
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5462.png

emd_5462_1.png

Downloads & links

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Map

FileDownload / File: emd_5462.map.gz / Format: CCP4 / Size: 89 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of soluble KNH1144-SOSIP Env trimers in complex with Fab 17b
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 520.0 / Movie #1: 520
Minimum - Maximum-291.433776860000023 - 1033.002563479999935
Average (Standard dev.)6.17095995 (±72.759666440000004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-144-144-144
Dimensions288288288
Spacing288288288
CellA=B=C: 311.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z311.040311.040311.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS-144-144-144
NC/NR/NS288288288
D min/max/mean-291.4341033.0036.171

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Supplemental data

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Sample components

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Entire : Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.

EntireName: Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.
Components
  • Sample: Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.
  • Protein or peptide: Envelope glycoprotein
  • Protein or peptide: Fab portion of monoclonal antibody 17b

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Supramolecule #1000: Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.

SupramoleculeName: Molecular structure of KNH1144 SOSIP gp140 with 17b Fab.
type: sample / ID: 1000 / Oligomeric state: trimer / Number unique components: 2
Molecular weightTheoretical: 670 KDa

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Macromolecule #1: Envelope glycoprotein

MacromoleculeName: Envelope glycoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: Env
Details: Membrane proximal external region of KNH1144 (Genbank accession no. AY736812) with the following residue substitutions A662E, S668N, and S676T
Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: HIV-1 isolate 00KE_KNH1144 / synonym: HIV-1
Molecular weightExperimental: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: SOSIP-PPI4 and furin-pcDNA3.1

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Macromolecule #2: Fab portion of monoclonal antibody 17b

MacromoleculeName: Fab portion of monoclonal antibody 17b / type: protein_or_peptide / ID: 2 / Name.synonym: 17b Fab / Details: Fab fragment / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 50 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.42 mg/mL
BufferpH: 7.5 / Details: TNE Buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA)
GridDetails: Protochips C-flat R 2/2, plasma cleaned
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Method: blot for 6 seconds, blot offset of -2, plunge into an ethane slurry cooled by liquid nitrogen

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 80 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 138888 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateAug 17, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3299 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 / Number subtomograms used: 39001

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Atomic model buiding 1

Initial modelPDB ID:

1gc1


Chain - #0 - Chain ID: G / Chain - #1 - Chain ID: H / Chain - #2 - Chain ID: L
SoftwareName: Chimera
DetailsProtocol: Rigid body. Automated fitting procedures
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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