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- EMDB-1800: Single particle cryo-electron microscopy analysis of CD4 bound HI... -

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Basic information

Entry
Database: EMDB / ID: EMD-1800
TitleSingle particle cryo-electron microscopy analysis of CD4 bound HIV-1 Env
Map dataThis is an image of a surface rendered CD4 bound HIV-1 Env
Sample
  • Sample: CD4 bound HIV-1 Env
  • Protein or peptide: gp160deltaCTSOS
  • Protein or peptide: soluble CD4 (2 domain)
KeywordsHIV-1 spike / Membrane Fusion / Structural Transition / Cryo-EM / Single Particle
Function / homologyinduction by virus of host cell-cell fusion / T-cell surface antigen CD4 / Human immunodeficiency virus 1, envelope glycoprotein Gp120 / viral envelope
Function and homology information
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 21.0 Å
AuthorsWu SR / Loving R / Lindqvist B / Hebert H / Koeck P / Sjoberg M / Garoff H
CitationJournal: AIDS Res Hum Retroviruses / Year: 1990
Title: Escherichia coli expression, purification, and biological activity of a truncated soluble CD4.
Authors: R L Garlick / R J Kirschner / F M Eckenrode / W G Tarpley / C S Tomich /
Abstract: A truncated molecule containing the N-terminal 183 amino acid residues of CD4 (sCD4-183) has been produced in Escherichia coli at high levels, using the trp promoter and an AT-rich ribosome binding ...A truncated molecule containing the N-terminal 183 amino acid residues of CD4 (sCD4-183) has been produced in Escherichia coli at high levels, using the trp promoter and an AT-rich ribosome binding site to direct expression in a pBR322-derived vector. A culture has been selected which allows large-scale fermentation and production of this material as an insoluble inclusion body protein. Procedures which solubilize, refold, and purify sCD4-183 have been developed. The purified sCD4-183 binds gp120 in solution and blocks human immunodeficiency virus (HIV) infection of human peripheral blood lymphocytes in vitro.
History
DepositionSep 29, 2010-
Header (metadata) releaseOct 13, 2010-
Map releaseOct 29, 2010-
UpdateMay 24, 2012-
Current statusMay 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_1800.jpg

Downloads & links

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Map

FileDownload / File: emd_1800.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 CD4 bound HIV-1 Env
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 2.3 / Movie #1: 2.6
Minimum - Maximum-3.59058 - 5.97472
Average (Standard dev.)0.0786333 (±1.02616)
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-3.5915.9750.079

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Supplemental data

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Sample components

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Entire : CD4 bound HIV-1 Env

EntireName: CD4 bound HIV-1 Env
Components
  • Sample: CD4 bound HIV-1 Env
  • Protein or peptide: gp160deltaCTSOS
  • Protein or peptide: soluble CD4 (2 domain)

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Supramolecule #1000: CD4 bound HIV-1 Env

SupramoleculeName: CD4 bound HIV-1 Env / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: Trimer / Number unique components: 2
Molecular weightExperimental: 700 KDa / Theoretical: 500 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 weightTheoretical: 420 KDa
Recombinant expressionOrganism: 293T / Recombinant plasmid: pCAGGS JRFL 160deltaCTSOS
SequenceGO: viral envelope
InterPro: Human immunodeficiency virus 1, envelope glycoprotein Gp120

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Macromolecule #2: soluble CD4 (2 domain)

MacromoleculeName: soluble CD4 (2 domain) / type: protein_or_peptide / ID: 2 / Name.synonym: sCD4-2d
Details: sCD4-183 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 26 KDa
SequenceGO: induction by virus of host cell-cell fusion / InterPro: T-cell surface antigen CD4

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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 once 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
DateOct 23, 2009
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 3.5 µm / Number real images: 336 / 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: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 9870
DetailsThe 3D structures reconstructed by processing particle images using standard single particle procedure in EMAN version 1.9.

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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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