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- EMDB-5707: Negative stained image reconstruction of HIV KNH11444 subtype A S... -

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Basic information

Entry
Database: EMDB / ID: EMD-5707
TitleNegative stained image reconstruction of HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
Map dataNegative stained image reconstruction of HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
Sample
  • Sample: HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
  • Protein or peptide: Human immunodeficiency virus Envelope protein
  • Protein or peptide: Fab fragment of VRC PG04 monoclonal antibody
KeywordsHuman immunodeficiency virus / antigen / vaccine development / neutralization / conformational change / micelles
Biological speciesHuman immunodeficiency virus / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 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: 4.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5707.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stained image reconstruction of HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 4.15 / Movie #1: 4.15
Minimum - Maximum-2.94080257 - 11.885675429999999
Average (Standard dev.)0.0 (±1.00000012)
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-2.94111.8860.000

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Supplemental data

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Sample components

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Entire : HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab frag...

EntireName: HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
Components
  • Sample: HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
  • Protein or peptide: Human immunodeficiency virus Envelope protein
  • Protein or peptide: Fab fragment of VRC PG04 monoclonal antibody

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Supramolecule #1000: HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab frag...

SupramoleculeName: HIV KNH11444 subtype A SOSIP.664.dV1V2V3 in complex with Fab fragment of VRC PG04 monoclonal antibody
type: sample / ID: 1000 / Details: The sample was monodisperse via SEC. / Oligomeric state: Trimer / Number unique components: 2
Molecular weightTheoretical: 528 KDa

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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 weightTheoretical: 393 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293S / Recombinant plasmid: pPI4

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Macromolecule #2: Fab fragment of VRC PG04 monoclonal antibody

MacromoleculeName: Fab fragment of VRC PG04 monoclonal antibody / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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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
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 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
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
DateFeb 25, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 10.9 µm / Number real images: 220 / Average electron dose: 16 e/Å2 / Details: Data collected on CCD / Bits/pixel: 16
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 512
Final angle assignmentDetails: SPIDER: theta 45 degrees, phi 45 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx/EMAN2
Details: Final maps were calculated from two averaged data sets.
Number images used: 11958
DetailsParticles were selected using DogPicker, reference free class averages determined using Sparx, and initial model generated using common-lines with EMAN2.

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