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- EMDB-6194: Negative stain electron microscopy of JRFL SOSIP liganded with VRC01 -

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Basic information

Entry
Database: EMDB / ID: EMD-6194
TitleNegative stain electron microscopy of JRFL SOSIP liganded with VRC01
Map data
SampleJRFL SOSIP liganded with VRC01:
JRFL SOSIP gp140 / VRC01
Biological speciesSimian-Human immunodeficiency virus / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 20 Å
AuthorsGuenaga J / de Val N / Ward AB / Wyatt RT
CitationJournal: 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.
History
Current status-Processing site: RCSB / Status: Released
DepositionNov 19, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseJan 21, 2015-
UpdateJan 21, 2015-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 23
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 23
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_6194.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.05 Å/pix.
x 160 pix.
= 328. Å
2.05 Å/pix.
x 160 pix.
= 328. Å
2.05 Å/pix.
x 160 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy EMDB: 20.899999999999999 / Movie #1: 23
Minimum - Maximum-35.499401089999999 - 90.522430420000006
Average (Standard dev.)0.55622959 (±6.34946108)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
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/NS000
NC/NR/NS160160160
D min/max/mean-35.49990.5220.556

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Supplemental data

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Sample components

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Entire JRFL SOSIP liganded with VRC01

EntireName: JRFL SOSIP liganded with VRC01 / Number of components: 2
Oligomeric State: One trimer of JRFL SOSIP binds 3 VRC01 molecules
MassTheoretical: 570 kDa / Experimental: 570 kDa / Measured by: Size exclusion chromatography (SEC)

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Component #1: protein, JRFL SOSIP gp140

ProteinName: JRFL SOSIP gp140 / Oligomeric Details: trimer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 570 kDa / Experimental: 570 kDa
SourceSpecies: Simian-Human immunodeficiency virus
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK 293F

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Component #2: protein, VRC01

ProteinName: VRC01 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 3
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.5 mg/mL / Buffer solution: 50 mM Tris-HCl, 150 mM NaCl / pH: 7.4
Support film400 Cu mesh grids, glow-discharged at 15 mA for 30 seconds
StainingGrids were stained for 30 seconds with 2% uranyl formate.
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI SPIRIT / Date: Mar 26, 2014
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 29.28 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 46000 X (nominal), 52000 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 750 - 1000 nm
Specimen HolderModel: OTHER / Tilt Angle: 0 - 40 °
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 192

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 22762
3D reconstructionSoftware: EMAN2, sparx / Resolution: 20 Å / Resolution method: FSC 0.5, gold-standard

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3J5M

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