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- EMDB-5723: Negative stain Electron Microscopy of Bg505 SOSIP.664 in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5723
TitleNegative stain Electron Microscopy of Bg505 SOSIP.664 in complex with sCD4 and 17b
Map dataReconstruction of BG505 SOSIP.664 in complex with sCD4 and Fab 17b
Sample
  • Sample: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
  • Protein or peptide: HIV spike protein BG505 SOSIP.664
  • Protein or peptide: sCD4
  • Protein or peptide: 17b Fab
KeywordsHIV vaccine / Env trimer / Bg505 SOSIP.664 / sCD4 / 17b
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / virus-mediated perturbation of host defense response / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / structural molecule activity / virion attachment to host cell / host cell plasma membrane / virion membrane / identical protein binding / plasma membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus / Escherichia coli (E. coli) / Cricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsSanders RW / Derking R / Cupo A / Julien JP / Yasmeen A / de Val N / Kim HJ / Blattner C / Torrents A / Korzun J ...Sanders RW / Derking R / Cupo A / Julien JP / Yasmeen A / de Val N / Kim HJ / Blattner C / Torrents A / Korzun J / Golabek M / de los Reyes K / Ketas TJ / van Gils MJ / King CR / Wilson IA / Ward AB / Klasse PJ / Moore JP
CitationJournal: PLoS Pathog / Year: 2013
Title: A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.
Authors: Rogier W Sanders / Ronald Derking / Albert Cupo / Jean-Philippe Julien / Anila Yasmeen / Natalia de Val / Helen J Kim / Claudia Blattner / Alba Torrents de la Peña / Jacob Korzun / Michael ...Authors: Rogier W Sanders / Ronald Derking / Albert Cupo / Jean-Philippe Julien / Anila Yasmeen / Natalia de Val / Helen J Kim / Claudia Blattner / Alba Torrents de la Peña / Jacob Korzun / Michael Golabek / Kevin de Los Reyes / Thomas J Ketas / Marit J van Gils / C Richter King / Ian A Wilson / Andrew B Ward / P J Klasse / John P Moore /
Abstract: A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the ...A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.
History
DepositionJul 24, 2013-
Header (metadata) releaseAug 28, 2013-
Map releaseOct 9, 2013-
UpdateOct 9, 2013-
Current statusOct 9, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5723_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5723.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of BG505 SOSIP.664 in complex with sCD4 and Fab 17b
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-5.03321362 - 16.132875439999999
Average (Standard dev.)0.00000001 (±0.91930789)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-47-47-47
Dimensions192192192
Spacing192192192
CellA=B=C: 393.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-47-47-47
NC/NR/NS192192192
D min/max/mean-5.03316.1330.000

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Supplemental data

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Sample components

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Entire : HIV spike protein 664G construct in complex with sCD4 and Fab fra...

EntireName: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
Components
  • Sample: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
  • Protein or peptide: HIV spike protein BG505 SOSIP.664
  • Protein or peptide: sCD4
  • Protein or peptide: 17b Fab

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Supramolecule #1000: HIV spike protein 664G construct in complex with sCD4 and Fab fra...

SupramoleculeName: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
type: sample / ID: 1000
Details: The sCD4 was incubated for 1h at 4C with BG505 SOSIP.664. After, this complex was incubated for 40 minutes at 4C with 17b. Finally, this complex was purified using size exclusion chromatography and concentrated.
Oligomeric state: one trimer of BG505 binds to three Fabs and three sCD4
Number unique components: 3
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa / Method: Size exclusion Chromatography

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Macromolecule #1: HIV spike protein BG505 SOSIP.664

MacromoleculeName: HIV spike protein BG505 SOSIP.664 / type: protein_or_peptide / ID: 1 / Name.synonym: BG505 SOSIP.664
Details: BG505 SOSIP.664 was complexed with sCD4 and Fab 17b
Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus / synonym: HIV
Molecular weightExperimental: 360 KDa / Theoretical: 360 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pPPI4
SequenceUniProtKB: Envelope glycoprotein gp160

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Macromolecule #2: sCD4

MacromoleculeName: sCD4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: 17b Fab

MacromoleculeName: 17b Fab / type: protein_or_peptide / ID: 3
Details: 17b Fab was expressed as an IgG and digested with papain to produce Fab.
Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Cricetulus griseus (Chinese hamster) / synonym: Chinese hamster
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: CHO

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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

Concentration0.03 mg/mL
BufferpH: 7.4 / Details: 50 mM TRIS-HCl, 150 mM NaCl
StainingType: NEGATIVE / Details: 2% w/v Uranyl Formate for 25 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: LAB6
Electron opticsCalibrated magnification: 52050 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 50
TemperatureMin: 292 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
DateJul 16, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 176 / Average electron dose: 30 e/Å2 / Od range: 1.4
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 128
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, EMAN1
Details: The particles were selected using a DoG Picker, and cleaned using reference-free class averaging. The final map was calculated from a single dataset.
Number images used: 22145
DetailsParticles were picked automatically using DoG Picker and put into a particle stack using the Appion software package. Initial, reference-free, two-dimensional (2D) class averages were calculated using particles binned by five via the Xmipp Clustering 2D Alignment and sorted into classes. EMAN was used for the 3D reconstruction.

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Atomic model buiding 1

Initial modelPDB ID:

1rzk

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation, Fit in Map, Chimera

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