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Yorodumi- EMDB-6256: A native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6256 | |||||||||
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Title | A native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene | |||||||||
Map data | B41 SOSIP with VRC01 | |||||||||
Sample |
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Biological species | Simian-Human immunodeficiency virus / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 17.0 Å | |||||||||
Authors | Pugach P / Ozorowski G | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6256.map.gz | 10 MB | EMDB map data format | |
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Header (meta data) | emd-6256-v30.xml emd-6256.xml | 10.5 KB 10.5 KB | Display Display | EMDB header |
Images | 400_6256.gif 80_6256.gif | 12.9 KB 1.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6256 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6256 | HTTPS FTP |
-Validation report
Summary document | emd_6256_validation.pdf.gz | 77.5 KB | Display | EMDB validaton report |
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Full document | emd_6256_full_validation.pdf.gz | 76.6 KB | Display | |
Data in XML | emd_6256_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6256 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6256 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6256.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | B41 SOSIP with VRC01 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : B41 SOSIP liganded with VRC01
Entire | Name: B41 SOSIP liganded with VRC01 |
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Components |
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-Supramolecule #1000: B41 SOSIP liganded with VRC01
Supramolecule | Name: B41 SOSIP liganded with VRC01 / type: sample / ID: 1000 / Oligomeric state: one B41 trimer bound to three Fabs / Number unique components: 2 |
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Molecular weight | Experimental: 570 KDa / Theoretical: 570 KDa / Method: Size Exclusion Chromatography (SEC) |
-Macromolecule #1: B41 SOSIP.664 gp140
Macromolecule | Name: B41 SOSIP.664 gp140 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes |
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Source (natural) | Organism: Simian-Human immunodeficiency virus / synonym: HIV |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pPPI4 |
-Macromolecule #2: antibody VRC01 Fab
Macromolecule | Name: antibody VRC01 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.4 / Details: 50 mM Tris-HCl, 150 mM NaCl |
Staining | Type: NEGATIVE / Details: 2% w/v uranyl formate for 60 seconds |
Grid | Details: carbon-coated 400 Cu mesh grid, glow-discharged at 20 mA for 30 seconds |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Date | Jul 7, 2014 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 167 / Average electron dose: 23.12 e/Å2 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 46000 |
Sample stage | Specimen holder model: OTHER / Tilt angle max: 50 |
Experimental equipment | Model: Tecnai Spirit / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: 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 model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |