+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6297 | |||||||||
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Title | JRFL gp140 NFL2 liganded to 2G12 | |||||||||
Map data | JRFL gp140 NFL liganded to 2G12 | |||||||||
Sample |
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Biological species | Human immunodeficiency virus 1 / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
Authors | de Val N / Sharma S / Bale S | |||||||||
Citation | Journal: Cell Rep / Year: 2015 Title: Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design. Authors: Shailendra Kumar Sharma / Natalia de Val / Shridhar Bale / Javier Guenaga / Karen Tran / Yu Feng / Viktoriya Dubrovskaya / Andrew B Ward / Richard T Wyatt / Abstract: Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, ...Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses. | |||||||||
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_6297.map.gz | 14.7 MB | EMDB map data format | |
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Header (meta data) | emd-6297-v30.xml emd-6297.xml | 9.9 KB 9.9 KB | Display Display | EMDB header |
Images | 400_6297.gif 80_6297.gif | 12.1 KB 1.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6297 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6297 | HTTPS FTP |
-Validation report
Summary document | emd_6297_validation.pdf.gz | 78.1 KB | Display | EMDB validaton report |
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Full document | emd_6297_full_validation.pdf.gz | 77.2 KB | Display | |
Data in XML | emd_6297_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6297 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6297 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6297.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | JRFL gp140 NFL liganded to 2G12 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : JRFL gp140 NFL liganded to 2G12
Entire | Name: JRFL gp140 NFL liganded to 2G12 |
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Components |
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-Supramolecule #1000: JRFL gp140 NFL liganded to 2G12
Supramolecule | Name: JRFL gp140 NFL liganded to 2G12 / type: sample / ID: 1000 / Oligomeric state: trimer / Number unique components: 2 |
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Molecular weight | Experimental: 720 KDa / Theoretical: 720 KDa / Method: Size exclusion chromatography (SEC) |
-Macromolecule #1: JRFL gp140 NFL
Macromolecule | Name: JRFL gp140 NFL / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes |
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Source (natural) | Organism: Human immunodeficiency virus 1 / synonym: HIV-1 |
Molecular weight | Experimental: 720 KDa / Theoretical: 720 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK 293T |
-Macromolecule #2: 2G12 Fab
Macromolecule | Name: 2G12 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% uranyl formate for 30 seconds |
Grid | Details: 400 mesh Cu grids, negatively glow discharged for 30 seconds |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Date | Feb 25, 2014 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 191 / Average electron dose: 28.15 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: 1.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 46000 |
Sample stage | Specimen holder model: HOME BUILD / Tilt angle max: 50 |
Experimental equipment | Model: Tecnai Spirit / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: EMAN, sparx / Number images used: 9311 |
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-Atomic model buiding 1
Initial model | PDB ID: |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT |