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- EMDB-6591: CSF trimer in a post-fusion or intermediate state -

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Basic information

Entry
Database: EMDB / ID: EMD-6591
TitleCSF trimer in a post-fusion or intermediate state
Map dataReconstruction of the CSF trimer in a post-fusion state
Sample
  • Sample: CSF trimer in a post-fusion or intermediate state
  • Protein or peptide: CSF
KeywordsCSF-envelope glycoprotein trimer HIV-1 Keywords: HIV / envelope / glycoprotein / trimer
Biological speciesHuman immunodeficiency virus
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsKong P / He L / de Val N / Morris CD / Vora N / Azadnia P / Sok D / Zhou B / Burton DR / Ward AB ...Kong P / He L / de Val N / Morris CD / Vora N / Azadnia P / Sok D / Zhou B / Burton DR / Ward AB / Wilson IA / Zhu J
CitationJournal: Nat Commun / Year: 2016
Title: Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability.
Authors: Leopold Kong / Linling He / Natalia de Val / Nemil Vora / Charles D Morris / Parisa Azadnia / Devin Sok / Bin Zhou / Dennis R Burton / Andrew B Ward / Ian A Wilson / Jiang Zhu /
Abstract: The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. ...The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.
History
DepositionFeb 11, 2016-
Header (metadata) releaseApr 27, 2016-
Map releaseJul 13, 2016-
UpdateAug 24, 2016-
Current statusAug 24, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 10.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6591.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the CSF trimer in a post-fusion state
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 10.1 / Movie #1: 10.1
Minimum - Maximum-11.509586329999999 - 100.873313899999999
Average (Standard dev.)0.1996555 (±4.83763313)
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-11.510100.8730.200

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Supplemental data

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Sample components

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Entire : CSF trimer in a post-fusion or intermediate state

EntireName: CSF trimer in a post-fusion or intermediate state
Components
  • Sample: CSF trimer in a post-fusion or intermediate state
  • Protein or peptide: CSF

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Supramolecule #1000: CSF trimer in a post-fusion or intermediate state

SupramoleculeName: CSF trimer in a post-fusion or intermediate state / type: sample / ID: 1000 / Oligomeric state: trimer / Number unique components: 1
Molecular weightTheoretical: 420 KDa

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Macromolecule #1: CSF

MacromoleculeName: CSF / type: protein_or_peptide / ID: 1
Details: Heterodimer of gp120 and gp41 assembles into a trimer.
Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Human immunodeficiency virus
Molecular weightExperimental: 420 KDa / Theoretical: 420 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293F

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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

Concentration0.02 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE
Details: Grids were glow-discharged at 20 mA for 30 seconds and stained with 2% uranyl formate for 40 seconds.
GridDetails: 400 Cu mesh grids
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: 1.0 µm / Nominal magnification: 46000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateAug 15, 2015
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 53 / Average electron dose: 29.58 e/Å2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 173
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: EMAN2, sparx / Number images used: 17664

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