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- EMDB-5700: Negative stained image reconstruction of HIV KNH11444 subtype A S... -

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Basic information

Entry
Database: EMDB / ID: EMD-5700
TitleNegative stained image reconstruction of HIV KNH11444 subtype A SOSIP.681
Map dataNegative stained image reconstruction of KNH1144 SOSIP.681
Sample
  • Sample: HIV SOSIP.681 from KNH1144 subtype A
  • Protein or peptide: Human immunodeficiency virus Envelope protein
KeywordsHuman immunodeficiency virus / antigen / vaccine development / neutralization / conformational change / micelles
Biological speciesHuman immunodeficiency virus
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 Å
AuthorsKhayat R / Lee JH / Wilson IA / Ward AB
CitationJournal: J Virol / Year: 2013
Title: Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein.
Authors: Per Johan Klasse / Rafael S Depetris / Robert Pejchal / Jean-Philippe Julien / Reza Khayat / Jeong Hyun Lee / Andre J Marozsan / Albert Cupo / Nicolette Cocco / Jacob Korzun / Anila Yasmeen ...Authors: Per Johan Klasse / Rafael S Depetris / Robert Pejchal / Jean-Philippe Julien / Reza Khayat / Jeong Hyun Lee / Andre J Marozsan / Albert Cupo / Nicolette Cocco / Jacob Korzun / Anila Yasmeen / Andrew B Ward / Ian A Wilson / Rogier W Sanders / John P Moore /
Abstract: We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as ...We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.
History
DepositionJun 26, 2013-
Header (metadata) releaseJul 3, 2013-
Map releaseJul 24, 2013-
UpdateAug 21, 2013-
Current statusAug 21, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5700.png

Downloads & links

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Map

FileDownload / File: emd_5700.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stained image reconstruction of KNH1144 SOSIP.681
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.18 Å/pix.
x 160 pix.
= 348.8 Å		2.18 Å/pix.
x 160 pix.
= 348.8 Å		2.18 Å/pix.
x 160 pix.
= 348.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.18 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.4 / Movie #1: 3.4
Minimum - Maximum-2.47214103 - 10.91848755
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-2.47210.9180.000

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Supplemental data

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Sample components

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Entire : HIV SOSIP.681 from KNH1144 subtype A

EntireName: HIV SOSIP.681 from KNH1144 subtype A
Components
  • Sample: HIV SOSIP.681 from KNH1144 subtype A
  • Protein or peptide: Human immunodeficiency virus Envelope protein

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Supramolecule #1000: HIV SOSIP.681 from KNH1144 subtype A

SupramoleculeName: HIV SOSIP.681 from KNH1144 subtype A / type: sample / ID: 1000 / Details: The sample was monodisperse via SEC. / Oligomeric state: Trimer / Number unique components: 1
Molecular weightExperimental: 480 KDa / Theoretical: 480 KDa / Method: Mass spectroscopy

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Macromolecule #1: Human immunodeficiency virus Envelope protein

MacromoleculeName: Human immunodeficiency virus Envelope protein / type: protein_or_peptide / ID: 1 / Name.synonym: gp120/gp41 / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus / Strain: KNH1144 / synonym: HIV
Molecular weightExperimental: 480 KDa / Theoretical: 480 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293S / Recombinant plasmid: pPI4

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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

Concentration0.1 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES, pH 7.5, 50 mM NaCl
StainingType: NEGATIVE / Details: 2% Uranyl Formate for 30 seconds
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 293 K / Max: 294 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Legacy - Electron beam tilt params: -2
DateOct 27, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 10.9 µm / Number real images: 180 / Average electron dose: 16 e/Å2 / Details: Data collected on CCD / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected using DogPicker, reference free class averages determined using Sparx, and initial model generated using common-lines with EMAN2.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx/EMAN2
Details: Final maps were calculated from three averaged data sets.
Number images used: 5543
Final angle assignmentDetails: SPIDER: theta 45 degrees, phi 45 degrees
Final two d classificationNumber classes: 32

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