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- EMDB-6293: JRFL gp140 NFL2P unliganded -

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Basic information

Entry
Database: EMDB / ID: EMD-6293
TitleJRFL gp140 NFL2P unliganded
Map dataReconstruction of JRFL gp140 NFL unliganded
Sample
  • Sample: JRFL gp140 NFL2P unliganded
  • Protein or peptide: JRFL gp140 NFL2
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 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: 3.73
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.73
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6293.gif

Downloads & links

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Map

FileDownload / File: emd_6293.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of JRFL gp140 NFL unliganded
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 AUTHOR: 3.73 / Movie #1: 3.73
Minimum - Maximum-1.3027091 - 14.41309547
Average (Standard dev.)0.0 (±0.8805297)
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
MAP C/R/S123
start NC/NR/NS-39-39-39
NC/NR/NS160160160
D min/max/mean-1.30314.4130.000

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Supplemental data

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Sample components

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Entire : JRFL gp140 NFL2P unliganded

EntireName: JRFL gp140 NFL2P unliganded
Components
  • Sample: JRFL gp140 NFL2P unliganded
  • Protein or peptide: JRFL gp140 NFL2

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Supramolecule #1000: JRFL gp140 NFL2P unliganded

SupramoleculeName: JRFL gp140 NFL2P unliganded / type: sample / ID: 1000 / Oligomeric state: trimer / Number unique components: 1
Molecular weightExperimental: 420 KDa / Theoretical: 420 KDa / Method: Size exclusion chromatography (SEC)

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Macromolecule #1: JRFL gp140 NFL2

MacromoleculeName: JRFL gp140 NFL2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus 1 / synonym: HIV-1
Molecular weightExperimental: 420 KDa / Theoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293T

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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
DateMay 6, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 163 / Average electron dose: 37.91 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: 0.7 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: HOME BUILD / Tilt angle max: 50
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: EMAN, sparx / Number images used: 16737

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Atomic model buiding 1

Initial modelPDB ID:

3j5m

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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