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- EMDB-4015: A 3.9 Angstrom structure of HIV-1 CA-SP1 assembled in presence of... -

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Basic information

Entry
Database: EMDB / ID: EMD-4015
TitleA 3.9 Angstrom structure of HIV-1 CA-SP1 assembled in presence of Bevirimat
Map data
Sample
  • Virus: Human immunodeficiency virus 1
    • Protein or peptide: Capsid protein p24
Function / homology
Function and homology information


viral budding via host ESCRT complex / ISG15 antiviral mechanism / host multivesicular body / viral nucleocapsid / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / RNA binding / zinc ion binding / membrane
Similarity search - Function
Gag protein p6 / Gag protein p6 / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein ...Gag protein p6 / Gag protein p6 / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 group M subtype B (isolate NY5) / Human immunodeficiency virus 1
Methodsubtomogram averaging / cryo EM / Resolution: 3.9 Å
AuthorsSchur FKM / Obr M / Hagen WJH / Wan W / Arjen JJ / Kirkpatrick JM / Sachse C / Kraeusslich H-G / Briggs JAG
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research FoundationBR 3635/2-1 Germany
German Research FoundationKR 906/7-1 Germany
CitationJournal: Science / Year: 2016
Title: An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.
Authors: Florian K M Schur / Martin Obr / Wim J H Hagen / William Wan / Arjen J Jakobi / Joanna M Kirkpatrick / Carsten Sachse / Hans-Georg Kräusslich / John A G Briggs /
Abstract: Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation ...Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1.
History
DepositionJun 9, 2016-
Header (metadata) releaseJun 29, 2016-
Map releaseJul 13, 2016-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.112
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.112
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5l93
  • Surface level: 0.112
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5l93
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4015.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.112 / Movie #1: 0.112
Minimum - Maximum-0.29335153 - 0.5368952
Average (Standard dev.)0.0006436251 (±0.04243836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2930.5370.001

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Supplemental data

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Sample components

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Entire : Human immunodeficiency virus 1

EntireName: Human immunodeficiency virus 1
Components
  • Virus: Human immunodeficiency virus 1
    • Protein or peptide: Capsid protein p24

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Supramolecule #1: Human immunodeficiency virus 1

SupramoleculeName: Human immunodeficiency virus 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Virus-like particles were obtained by in vitro assembly of a truncated Gag construct (deltaMACANCSP2) in presence of the maturation inhibitor Bevirimat
NCBI-ID: 11676 / Sci species name: Human immunodeficiency virus 1 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET11C

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Macromolecule #1: Capsid protein p24

MacromoleculeName: Capsid protein p24 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 group M subtype B (isolate NY5)
Strain: isolate NY5
Molecular weightTheoretical: 24.789396 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SPRTLNAWVK VVEEKAFSPE VIPMFSALSE GATPQDLNTM LNTVGGHQAA MQMLKETINE EAAEWDRLHP VHAGPIAPGQ MREPRGSDI AGTTSTLQEQ IGWMTHNPPI PVGEIYKRWI ILGLNKIVRM YSPTSILDIR QGPKEPFRDY VDRFYKTLRA E QASQEVKN ...String:
SPRTLNAWVK VVEEKAFSPE VIPMFSALSE GATPQDLNTM LNTVGGHQAA MQMLKETINE EAAEWDRLHP VHAGPIAPGQ MREPRGSDI AGTTSTLQEQ IGWMTHNPPI PVGEIYKRWI ILGLNKIVRM YSPTSILDIR QGPKEPFRDY VDRFYKTLRA E QASQEVKN WMTETLLVQN ANPDCKTILK ALGPGATLEE MMTACQGVGG PGHKARVLAE AMSQVT

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHEPES
100.0 mMSodium ChlorideNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
1.0 mMTCEP

Details: Virus-like particles were assembled in the presence of nucleic acid (73mer oligonucleotide, 1:10 molar ratio oligonucleotide:protein).
GridModel: C-flat / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: at 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 15 K / Instrument: FEI VITROBOT MARK II
Details: 10nM colloidal gold was added to the sample prior to plunge freezing..
DetailsVirus-like particles were assembled in vitro

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsNanoprobe
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 8-10 / Number grids imaged: 2 / Average exposure time: 1.0 sec. / Average electron dose: 3.4 e/Å2
Details: Number of frames ranged from 8-10 Exposure time per tilt ranged from 0.8 to 1.0 seconds
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 43 / Number images used: 527528
Details: Subtomograms were extracted from the surface of each particle according to the determined radius of the particle.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4)CTF determination
CTFPHASEFLIPCTF correction within IMOD

Details: CTF correction was performed using the ctfphaseflip program in IMOD prior to backprojection.
Final angle assignmentType: OTHER / Software: (Name: AV3, TOM Toolbox) / Details: Cross-correlation based template matching
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: AV3, TOM Toolbox) / Number subtomograms used: 128733
DetailsFrames were aligned using MotionCorr. Tilts in a tilt series were exposure filtered for cumulative electron dose. Tomograms were reconstructed using IMOD.

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Atomic model buiding 1

Initial model
PDB IDChain

chain_id: 1, residue_range: 148-279

chain_id: 1, residue_range: 280-353

chain_id: 2, residue_range: 354-371
RefinementProtocol: OTHER
Output model

PDB-5l93:
An atomic model of HIV-1 CA-SP1 reveals structures regulating assembly and maturation

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