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- EMDB-6199: Negative stain electron microscopy of JRFL SOSIP liganded with PG... -

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Basic information

Entry
Database: EMDB / ID: EMD-6199
TitleNegative stain electron microscopy of JRFL SOSIP liganded with PGV04 at day 7
Map dataReconstruction of JRFL SOSIP liganded with PGV04 at day 7 (stability test)
Sample
  • Sample: JRFL SOSIP liganded with PGV04 at day 7 (stability test)
  • Protein or peptide: JRFL SOSIP gp140
  • Protein or peptide: PGV04
Biological speciesSimian-Human immunodeficiency virus / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 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
DepositionNov 20, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseJan 21, 2015-
UpdateJan 21, 2015-
Current statusJan 21, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6199.gif

Downloads & links

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Map

FileDownload / File: emd_6199.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of JRFL SOSIP liganded with PGV04 at day 7 (stability test)
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

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 4.8 / Movie #1: 3
Minimum - Maximum-3.37899542 - 13.368928909999999
Average (Standard dev.)0.00000001 (±0.8824873)
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-3.37913.3690.000

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Supplemental data

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Sample components

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Entire : JRFL SOSIP liganded with PGV04 at day 7 (stability test)

EntireName: JRFL SOSIP liganded with PGV04 at day 7 (stability test)
Components
  • Sample: JRFL SOSIP liganded with PGV04 at day 7 (stability test)
  • Protein or peptide: JRFL SOSIP gp140
  • Protein or peptide: PGV04

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Supramolecule #1000: JRFL SOSIP liganded with PGV04 at day 7 (stability test)

SupramoleculeName: JRFL SOSIP liganded with PGV04 at day 7 (stability test)
type: sample / ID: 1000
Oligomeric state: One trimer of JRFL SOSIP binds 3 PGV04 molecules
Number unique components: 2
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa / Method: Size exclusion chromatography (SEC)

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Macromolecule #1: JRFL SOSIP gp140

MacromoleculeName: JRFL SOSIP gp140 / type: protein_or_peptide / ID: 1 / Details: Complex was analyzed at day 7 for stability test. / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Simian-Human immunodeficiency virus
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F

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Macromolecule #2: PGV04

MacromoleculeName: PGV04 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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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

Concentration0.5 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE
Details: Grids were stained for 30 seconds with 2% uranyl formate.
GridDetails: 400 Cu mesh grids, glow-discharged at 15 mA for 30 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
DateFeb 14, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 206 / Average electron dose: 32.24 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 50
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: EMAN2, sparx / Number images used: 35657

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Atomic model buiding 1

Initial modelPDB ID:

3j5m

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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