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- EMDB-8268: HIV NFL trimer cross-linked with Glutaraldehyde and in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-8268
TitleHIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01
Map dataHIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01
Sample
  • Complex: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsWard AB / de Val N
CitationJournal: PLoS Pathog / Year: 2016
Title: Thermostability of Well-Ordered HIV Spikes Correlates with the Elicitation of Autologous Tier 2 Neutralizing Antibodies.
Authors: Yu Feng / Karen Tran / Shridhar Bale / Shailendra Kumar / Javier Guenaga / Richard Wilson / Natalia de Val / Heather Arendt / Joanne DeStefano / Andrew B Ward / Richard T Wyatt /
Abstract: In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more ...In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were "stress-tested" at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.
History
DepositionJun 28, 2016-
Header (metadata) releaseJul 13, 2016-
Map releaseJul 13, 2016-
UpdateApr 11, 2018-
Current statusApr 11, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 37.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 37.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8268.png

Downloads & links

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Map

FileDownload / File: emd_8268.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV NFL trimer cross-linked with Glutaraldehyde and in complex with bnAb VRC01
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 80 pix.
= 328. Å		4.1 Å/pix.
x 80 pix.
= 328. Å		4.1 Å/pix.
x 80 pix.
= 328. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 37.299999999999997 / Movie #1: 37.3
Minimum - Maximum-64.760859999999994 - 142.687440000000009
Average (Standard dev.)0.48541144 (±10.403214999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-64.761142.6870.485

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Supplemental data

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Sample components

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Entire : BG505_NFL_trimer cross-linked with glutaraldehyde and in complex ...

EntireName: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb
Components
  • Complex: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb

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Supramolecule #1: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex ...

SupramoleculeName: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with VRC01 bnAb
type: complex / ID: 1 / Parent: 0
Details: BG505_NFL_trimer cross-linked with glutaraldehyde and in complex with 3 broadly neutralizing antibodies VRC01
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: pPPI4
Molecular weightTheoretical: 570 KDa

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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

Concentration0.01 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMC4H12ClNO3Tris-HCl
50.0 mMNaClsodium chloride
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample was stained using two applications of 3 uL 2% UF.
GridModel: Electron Microscopy Sciences / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Details: The grids were glow-discharged at 20 mA.
DetailsThe trimer was incubated at RT with a 10 M excess of the broadly neutralizing antibody VRC01. The complex was purified by SEC.

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 60 / Average electron dose: 36.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 1.0 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 60000
Final reconstructionNumber classes used: 100 / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX / Number images used: 10234
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 100 / Avg.num./class: 50 / Software - Name: MRA/MSA

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