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- EMDB-6256: A native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene -

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Basic information

Entry
Database: EMDB / ID: EMD-6256
TitleA native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene
Map dataB41 SOSIP with VRC01
Sample
  • Sample: B41 SOSIP liganded with VRC01
  • Protein or peptide: B41 SOSIP.664 gp140
  • Protein or peptide: antibody VRC01 Fab
Biological speciesSimian-Human immunodeficiency virus / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsPugach P / Ozorowski G
CitationJournal: J Virol / Year: 2015
Title: A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.
Authors: Pavel Pugach / Gabriel Ozorowski / Albert Cupo / Rajesh Ringe / Anila Yasmeen / Natalia de Val / Ronald Derking / Helen J Kim / Jacob Korzun / Michael Golabek / Kevin de Los Reyes / Thomas J ...Authors: Pavel Pugach / Gabriel Ozorowski / Albert Cupo / Rajesh Ringe / Anila Yasmeen / Natalia de Val / Ronald Derking / Helen J Kim / Jacob Korzun / Michael Golabek / Kevin de Los Reyes / Thomas J Ketas / Jean-Philippe Julien / Dennis R Burton / Ian A Wilson / Rogier W Sanders / P J Klasse / Andrew B Ward / John P Moore /
Abstract: Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) ...Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.
IMPORTANCE: The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For ...IMPORTANCE: The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.
History
DepositionJan 23, 2015-
Header (metadata) releaseFeb 4, 2015-
Map releaseFeb 4, 2015-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.47
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.47
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6256.gif

Downloads & links

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Map

FileDownload / File: emd_6256.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB41 SOSIP with VRC01
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 3.47 / Movie #1: 3.47
Minimum - Maximum-2.01872325 - 11.828563689999999
Average (Standard dev.)-0.00000001 (±0.93446207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-39-39-39
Dimensions160160160
Spacing160160160
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS-39-39-39
NC/NR/NS160160160
D min/max/mean-2.01911.829-0.000

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Supplemental data

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Sample components

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Entire : B41 SOSIP liganded with VRC01

EntireName: B41 SOSIP liganded with VRC01
Components
  • Sample: B41 SOSIP liganded with VRC01
  • Protein or peptide: B41 SOSIP.664 gp140
  • Protein or peptide: antibody VRC01 Fab

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Supramolecule #1000: B41 SOSIP liganded with VRC01

SupramoleculeName: B41 SOSIP liganded with VRC01 / type: sample / ID: 1000 / Oligomeric state: one B41 trimer bound to three Fabs / Number unique components: 2
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa / Method: Size Exclusion Chromatography (SEC)

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Macromolecule #1: B41 SOSIP.664 gp140

MacromoleculeName: B41 SOSIP.664 gp140 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Simian-Human immunodeficiency virus / synonym: HIV
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pPPI4

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Macromolecule #2: antibody VRC01 Fab

MacromoleculeName: antibody VRC01 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE / Details: 2% w/v uranyl formate for 60 seconds
GridDetails: carbon-coated 400 Cu mesh grid, glow-discharged at 20 mA for 30 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 50
DateJul 7, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 167 / Average electron dose: 23.12 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: Sparx, EMAN2 / Number images used: 32943

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Atomic model buiding 1

Initial modelPDB ID:

3j5m

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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