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- EMDB-6296: Bg505 gp140 NFL2P liganded to PGV04 -

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Basic information

Entry
Database: EMDB / ID: EMD-6296
TitleBg505 gp140 NFL2P liganded to PGV04
Map dataBg505 gp140 NFL2 liganded to PGV04
Sample
  • Sample: Bg505 gp140 NFL liganded to PGV04
  • Protein or peptide: Bg505 gp140 NFL2P
  • Protein or peptide: PGV04 Fab
Biological speciesHuman immunodeficiency virus 1 / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
Authorsde Val N / Sharma S / Bale S
CitationJournal: 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
DepositionMar 10, 2015-
Header (metadata) releaseMar 25, 2015-
Map releaseMar 25, 2015-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 32.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 32.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6296.gif

Downloads & links

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Map

FileDownload / File: emd_6296.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBg505 gp140 NFL2 liganded to PGV04
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 32.700000000000003 / Movie #1: 32.7
Minimum - Maximum-84.726364140000001 - 165.809158330000002
Average (Standard dev.)0.57261777 (±10.576905249999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
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
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-84.726165.8090.573

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Supplemental data

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Sample components

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Entire : Bg505 gp140 NFL liganded to PGV04

EntireName: Bg505 gp140 NFL liganded to PGV04
Components
  • Sample: Bg505 gp140 NFL liganded to PGV04
  • Protein or peptide: Bg505 gp140 NFL2P
  • Protein or peptide: PGV04 Fab

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Supramolecule #1000: Bg505 gp140 NFL liganded to PGV04

SupramoleculeName: Bg505 gp140 NFL liganded to PGV04 / type: sample / ID: 1000 / Oligomeric state: trimer / Number unique components: 2
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa / Method: Size exclusion chromatography (SEC)

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Macromolecule #1: Bg505 gp140 NFL2P

MacromoleculeName: Bg505 gp140 NFL2P / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / synonym: HIV-1
Molecular weightExperimental: 570 KDa / Theoretical: 570 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T

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Macromolecule #2: PGV04 Fab

MacromoleculeName: PGV04 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human

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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 / Details: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE / Details: 2% uranyl formate for 30 seconds
GridDetails: 400 mesh Cu grids, negatively glow discharged for 30 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: HOME BUILD / Tilt angle max: 50
DateMay 16, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 131 / Average electron dose: 40.30 e/Å2
Tilt angle min0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: EMAN, sparx / Number images used: 28143

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Atomic model buiding 1

Initial modelPDB ID:

3j5m

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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