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- EMDB-8403: sub-tomogram average of in vitro assembled HIV-1 Gag VLPs -

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Basic information

Entry
Database: EMDB / ID: EMD-8403
Titlesub-tomogram average of in vitro assembled HIV-1 Gag VLPs
Map datasub-Tomogram average of in vitro reconstituted HIV-1 virus-like particles imaged prior to protease treatement confirming immature conformation of the Gag lattice.
Sample
  • Complex: gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6)Group-specific antigen
  • Protein or peptide: HIV-1 Gag VLPs
Biological speciesHuman immunodeficiency virus 1
Methodsubtomogram averaging / cryo EM / Resolution: 18.0 Å
AuthorsHimes BA / Zhang P
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM085043 United States
CitationJournal: Nat Commun / Year: 2016
Title: In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway.
Authors: Jiying Ning / Gonca Erdemci-Tandogan / Ernest L Yufenyuy / Jef Wagner / Benjamin A Himes / Gongpu Zhao / Christopher Aiken / Roya Zandi / Peijun Zhang /
Abstract: HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. ...HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.
History
Header (metadata) releaseOct 12, 2016-
DepositionOct 18, 2016-
Map releaseDec 21, 2016-
UpdateFeb 3, 2021-
Current statusFeb 3, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8403.png

Downloads & links

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Map

FileDownload / File: emd_8403.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsub-Tomogram average of in vitro reconstituted HIV-1 virus-like particles imaged prior to protease treatement confirming immature conformation of the Gag lattice.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.62 Å/pix.
x 120 pix.
= 434.4 Å		3.62 Å/pix.
x 120 pix.
= 434.4 Å		3.62 Å/pix.
x 120 pix.
= 434.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-17.132729 - 15.400311
Average (Standard dev.)-0.0014388983 (±0.99564207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 434.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.623.623.62
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z434.400434.400434.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-210-210-210
NX/NY/NZ420420420
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-17.13315.400-0.001

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Supplemental data

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Half map: gold-standard half map used for FSC calculation, masked...

Fileemd_8403_half_map_1.map
Annotationgold-standard half map used for FSC calculation, masked by soft cylinder.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: gold-standard half map used for FSC calculation, masked...

Fileemd_8403_half_map_2.map
Annotationgold-standard half map used for FSC calculation, masked by soft cylinder.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6)

EntireName: gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6)Group-specific antigen
Components
  • Complex: gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6)Group-specific antigen
  • Protein or peptide: HIV-1 Gag VLPs

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Supramolecule #1: gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6)

SupramoleculeName: gag polyprotein, Pr55gag ([delta]-MA15-100[delta]p6) / type: complex / ID: 1 / Parent: 0
Details: PRR-55 construct, expressed recombinantly without protease.
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta2 (De3) / Recombinant plasmid: prr400
Molecular weightTheoretical: 39.9 KDa

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Macromolecule #1: HIV-1 Gag VLPs

MacromoleculeName: HIV-1 Gag VLPs / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: GARASVLSGG ELDRALDKIE EEQNKSKKKA QQAAADTGHS SQVSQNYPIV QNIQGQMVHQ AISPRTLNAW VKVVEEKAFS PEVIPMFSAL SEGATPQDLN TMLNTVGGHQ AAMQMLKETI NEEAAEWDRV HPVHAGPIAP GQMREPRGSD IAGTTSTLQE QIGWMTNNPP ...String:
GARASVLSGG ELDRALDKIE EEQNKSKKKA QQAAADTGHS SQVSQNYPIV QNIQGQMVHQ AISPRTLNAW VKVVEEKAFS PEVIPMFSAL SEGATPQDLN TMLNTVGGHQ AAMQMLKETI NEEAAEWDRV HPVHAGPIAP GQMREPRGSD IAGTTSTLQE QIGWMTNNPP IPVGEIYKRW IILGLNKIVR MYSPTSILDI RQGPKEPFRD YVDRFYKTLR AEQASQEVKN WMTETLLVQN ANPDCKTILK ALGPAATLEE MMTACQGVGG PGHKARVLAE AMSQVTNTAT IMMQRGNFRN QRKMVKCFNC GKEGHTARNC RAPRKKGCWK CGKEGHQMKD CTERQANFLG KIWPSYKGRP

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

BufferpH: 6
Details: 50 mM sodium acetate (pH6.0), 100 microM ZnSO4 and 5 mM DTT
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER
DetailsThe sample forms a 3-dimensional lattice with ~8nm spacing. The sub-tomogram averages are aligned along the c6 symmetry axis, such that ~ 12 assymetric units are included for a total effective mass of ~ 0.45 MDa

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 77348 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 59000
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 28 / Number images used: 8400 / Reference model: EMD-2706 / Method: Cross-correlation based template matching / Software - Name: matlab scripts
CTF correctionSoftware - Name: matlab scripts
Details: Phase flipping on the projections, amplitude correction applied to final average.
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: matlab scripts / Number subtomograms used: 4000

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