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- EMDB-1801: Single particle cryo-electron microscopy analysis of unliganded H... -

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Basic information

Entry
Database: EMDB / ID: EMD-1801
TitleSingle particle cryo-electron microscopy analysis of unliganded HIV-1 Env
Map dataThis is an image of a surface rendered unliganded HIV-1 Env
Sample
  • Sample: Unliganded HIV-1 Env
  • Protein or peptide: gp160deltaCTSOS
KeywordsHIV-1 spike / Membrane Fusion / Structural Transition / Cryo-EM / Single Particle
Function / homologyHuman immunodeficiency virus 1, envelope glycoprotein Gp120 / viral envelope
Function and homology information
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 18.0 Å
AuthorsWu SR / Loving R / Lindqvist B / Hebert H / Koeck P / Sjoberg M / Garoff H
CitationJournal: Proc Natl Acad Sci U S A / Year: 2010
Title: Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure.
Authors: Shang-Rung Wu / Robin Löving / Birgitta Lindqvist / Hans Hebert / Philip J B Koeck / Mathilda Sjöberg / Henrik Garoff /
Abstract: The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. ...The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.
History
DepositionSep 29, 2010-
Header (metadata) releaseOct 8, 2010-
Map releaseOct 29, 2010-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_1801.jpg

Downloads & links

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Map

FileDownload / File: emd_1801.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of a surface rendered unliganded HIV-1 Env
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 2.8 / Movie #1: 2.8
Minimum - Maximum-4.92636 - 7.24269
Average (Standard dev.)0.0606024 (±1.00079)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 252 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-4.9267.2430.061

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Supplemental data

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Sample components

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Entire : Unliganded HIV-1 Env

EntireName: Unliganded HIV-1 Env
Components
  • Sample: Unliganded HIV-1 Env
  • Protein or peptide: gp160deltaCTSOS

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Supramolecule #1000: Unliganded HIV-1 Env

SupramoleculeName: Unliganded HIV-1 Env / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: Trimer / Number unique components: 1
Molecular weightExperimental: 550 KDa / Theoretical: 420 KDa / Method: Blue Native PAGE

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Macromolecule #1: gp160deltaCTSOS

MacromoleculeName: gp160deltaCTSOS / type: protein_or_peptide / ID: 1 / Name.synonym: HIV-1 Env / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: JR-FL / synonym: HIV-1
Molecular weightExperimental: 550 KDa / Theoretical: 420 KDa
Recombinant expressionOrganism: 293T / Recombinant plasmid: p C AGGS JRFL 160deltaCTSOS
SequenceGO: viral envelope
InterPro: Human immunodeficiency virus 1, envelope glycoprotein Gp120

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 50 mM HEPES, 100 mM NaCl, 1.8 mM CaCl2
StainingType: NEGATIVE / Details: No stain was applied
GridDetails: 200 mesh Cu Holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 77 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 43300
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 96 K / Average: 95 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using online FFT
Legacy - Electron beam tilt params: No Tilt
DateNov 18, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 3.5 µm / Number real images: 148 / Average electron dose: 9 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each Digitized Image
Final two d classificationNumber classes: 131
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 11213

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Atomic model buiding 1

Initial modelPDB ID:

3jwd


Chain - Chain ID: A
SoftwareName: O
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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