|Entry||Database: EMDB / ID: EMD-6194|
|Title||Negative stain electron microscopy of JRFL SOSIP liganded with VRC01|
|Sample||JRFL SOSIP liganded with VRC01:|
JRFL SOSIP gp140 / VRC01
|Biological species||Simian-Human immunodeficiency virus / unidentified (others)|
|Method||single particle reconstruction / negative staining / Resolution: 20 Å|
|Authors||Guenaga J / de Val N / Ward AB / Wyatt RT|
|Citation||Journal: PLoS Pathog. / Year: 2015|
Title: Well-ordered trimeric HIV-1 subtype B and C soluble spike mimetics generated by negative selection display native-like properties.
Authors: Javier Guenaga / Natalia de Val / Karen Tran / Yu Feng / Karen Satchwell / Andrew B Ward / Richard T Wyatt /
Abstract: The structure of BG505 gp140 SOSIP, a soluble mimic of the native HIV-1 envelope glycoprotein (Env), marks the beginning of new era in Env structure-based immunogen design. Displaying a well-ordered ...The structure of BG505 gp140 SOSIP, a soluble mimic of the native HIV-1 envelope glycoprotein (Env), marks the beginning of new era in Env structure-based immunogen design. Displaying a well-ordered quaternary structure, these subtype A-derived trimers display an excellent antigenic profile, discriminating recognition by broadly neutralizing antibodies (bNAbs) from non-broadly neutralizing antibodies (non-bNAbs), and provide a solid Env-based immunogenic platform starting point. Even with this important advance, obtaining homogeneous well-ordered soluble SOSIP trimers derived from other subtypes remains challenging. Here, we report the "rescue" of homogeneous well-ordered subtype B and C SOSIP trimers from a heterogeneous Env mixture using CD4 binding site-directed (CD4bs) non-bNAbs in a negative-selection purification process. These non-bNAbs recognize the primary receptor CD4bs only on disordered trimers but not on the native Env spike or well-ordered soluble trimers due to steric hindrance. Following negative selection to remove disordered oligomers, we demonstrated recovery of well-ordered, homogeneous trimers by electron microscopy (EM). We obtained 3D EM reconstructions of unliganded trimers, as well as in complex with sCD4, a panel of CD4bs-directed bNAbs, and the cleavage-dependent, trimer-specific bNAb, PGT151. Using bio-layer light interferometry (BLI) we demonstrated that the well-ordered trimers were efficiently recognized by bNAbs and poorly recognized by non-bNAbs, representing soluble mimics of the native viral spike. Biophysical characterization was consistent with the thermostability of a homogeneous species that could be further stabilized by specific bNAbs. This study revealed that Env trimers generate different frequencies of well-ordered versus disordered aberrant trimers even when they are genetically identical. By negatively selecting the native-like well-ordered trimers, we establish a new means to obtain soluble Env mimetics derived from subtypes B and C for expanded use as candidate vaccine immunogens.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_6194.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.05 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire JRFL SOSIP liganded with VRC01
|Entire||Name: JRFL SOSIP liganded with VRC01 / Number of components: 2 |
Oligomeric State: One trimer of JRFL SOSIP binds 3 VRC01 molecules
|Mass||Theoretical: 570 kDa / Experimental: 570 kDa / Measured by: Size exclusion chromatography (SEC)|
-Component #1: protein, JRFL SOSIP gp140
|Protein||Name: JRFL SOSIP gp140 / Oligomeric Details: trimer / Number of Copies: 1 / Recombinant expression: Yes|
|Mass||Theoretical: 570 kDa / Experimental: 570 kDa|
|Source||Species: Simian-Human immunodeficiency virus|
|Source (engineered)||Expression System: Homo sapiens (human) / Cell of expression system: HEK 293F|
-Component #2: protein, VRC01
|Protein||Name: VRC01 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 3|
|Source||Species: unidentified (others)|
|Specimen||Specimen state: Particle / Method: negative staining|
|Sample solution||Specimen conc.: 0.5 mg/mL / Buffer solution: 50 mM Tris-HCl, 150 mM NaCl / pH: 7.4|
|Support film||400 Cu mesh grids, glow-discharged at 15 mA for 30 seconds|
|Staining||Grids were stained for 30 seconds with 2% uranyl formate.|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
Model: Tecnai Spirit / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI SPIRIT / Date: Mar 26, 2014|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 29.28 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 46000 X (nominal), 52000 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 750 - 1000 nm|
|Specimen Holder||Model: OTHER / Tilt Angle: 0 - 40 °|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Image acquisition||Number of digital images: 192|
|Processing||Method: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 22762|
|3D reconstruction||Software: EMAN2, sparx / Resolution: 20 Å / Resolution method: FSC 0.5, gold-standard|
-Atomic model buiding
|Modeling #1||Refinement protocol: rigid body / Refinement space: REAL|
Input PDB model: 3J5M
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