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- EMDB-2484: Pre-fusion structure of trimeric HIV-1 envelope glycoprotein dete... -

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Basic information

Entry
Database: EMDB / ID: EMD-2484
TitlePre-fusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy
Map dataStructure of HIV-1 gp140 trimer bound to VRC03 Fab
Sample
  • Sample: Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab.
  • Protein or peptide: gp120Envelope glycoprotein GP120
  • Protein or peptide: gp41
  • Protein or peptide: Monoclonal antibody VRC03 Fab Heavy chain
  • Protein or peptide: Monoclonal antibody VRC03 Fab Light chain
KeywordsHIV-1 envelope glycoprotein / SOSIP gp140 trimer / HIV-1 Env / pre-fusion state
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsBartesaghi A / Merk A / Borgnia MJ / Milne JLS / Subramaniam S
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy.
Authors: Alberto Bartesaghi / Alan Merk / Mario J Borgnia / Jacqueline L S Milne / Sriram Subramaniam /
Abstract: The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to ...The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target-cell membranes. Here, we present the cryo-EM structure, at subnanometer resolution (~6 Å at 0.143 FSC), of the 'closed', prefusion state of trimeric HIV-1 Env complexed to the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the 'open' quaternary conformation. The architecture of trimeric HIV-1 Env in the prefusion state and in the activated intermediate state resembles the corresponding states of influenza hemagglutinin trimers, thus providing direct evidence for the similarity in entry mechanisms used by HIV-1, influenza and related enveloped viruses.
History
DepositionOct 11, 2013-
Header (metadata) releaseOct 30, 2013-
Map releaseOct 30, 2013-
UpdateApr 16, 2014-
Current statusApr 16, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cc8
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2484_squ...

Downloads & links

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Map

FileDownload / File: emd_2484.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of HIV-1 gp140 trimer bound to VRC03 Fab
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0405 / Movie #1: 0.0405
Minimum - Maximum-0.03569165 - 0.08147071
Average (Standard dev.)0.00132176 (±0.00755302)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0360.0810.001

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Supplemental data

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Sample components

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Entire : Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab.

EntireName: Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab.
Components
  • Sample: Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab.
  • Protein or peptide: gp120Envelope glycoprotein GP120
  • Protein or peptide: gp41
  • Protein or peptide: Monoclonal antibody VRC03 Fab Heavy chain
  • Protein or peptide: Monoclonal antibody VRC03 Fab Light chain

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Supramolecule #1000: Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab.

SupramoleculeName: Complex of KNH1144 SOSIP gp140 trimer with VRC03 Fab. / type: sample / ID: 1000
Oligomeric state: Tetramer (gp41, gp120, Fab Heavy, Fab Light)
Number unique components: 4
Molecular weightTheoretical: 670 KDa

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Macromolecule #1: gp120

MacromoleculeName: gp120 / type: protein_or_peptide / ID: 1 / Name.synonym: Envelope glycoprotein / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: HIV-1 isolate 00KE_KNH1144 / synonym: HIV-1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: SOSIP-PPI4 and furin-pcDNA3.1

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Macromolecule #2: gp41

MacromoleculeName: gp41 / type: protein_or_peptide / ID: 2 / Name.synonym: Envelope Glycoprotein
Details: Complete ectodomain of HIV-1 Env from the Clade A strain KNH1144 inlcuding residues in the membrane proximal external region with the following residue substitutions A662E, S668N, and S676T
Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: HIV-1 isolate 00KE_KNH1144 / synonym: HIV-1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant plasmid: SOSIP-PPI4 and furin-pcDNA3.1

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Macromolecule #3: Monoclonal antibody VRC03 Fab Heavy chain

MacromoleculeName: Monoclonal antibody VRC03 Fab Heavy chain / type: protein_or_peptide / ID: 3 / Name.synonym: VRC03 Fab / Details: Fab fragment / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293

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Macromolecule #4: Monoclonal antibody VRC03 Fab Light chain

MacromoleculeName: Monoclonal antibody VRC03 Fab Light chain / type: protein_or_peptide / ID: 4 / Name.synonym: VRC03 Fab / Details: Fab fragment / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.65 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 IV
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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateAug 9, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 4713 / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 500
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN, IMAGIC, FREALIGN / Number images used: 88125
DetailsParticles were selected manually, classified in 2D using IMAGIC and 3D refinement was done with FREALIGN.

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Atomic model buiding 1

Initial modelPDB ID:

3se8


Chain - #0 - Chain ID: G / Chain - #1 - Chain ID: H / Chain - #2 - Chain ID: L
SoftwareName: Chimera
Details3SE8, Chain G and chains H, L fitted as two separate rigid bodies into map. 3HMG helix fitted by hand into map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4cc8:
Pre-fusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy

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Atomic model buiding 2

Initial modelPDB ID:

3hmg


Chain - Chain ID: B
SoftwareName: Chimera
DetailsProtocol: Rigid body. Automated fitting procedures. Only residues 92-122 of chain B were fitted.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4cc8:
Pre-fusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy

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