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- PDB-5l93: An atomic model of HIV-1 CA-SP1 reveals structures regulating ass... -

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Basic information

Entry
Database: PDB / ID: 5l93
TitleAn atomic model of HIV-1 CA-SP1 reveals structures regulating assembly and maturation
ComponentsCapsid protein p24
KeywordsVIRAL PROTEIN / HIV-1 capsid SP1
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
Retrovirus capsid C-terminal domain / Human Immunodeficiency Virus Type 1 Capsid Protein / Human Immunodeficiency Virus Type 1 Capsid Protein / Gag protein p6 / Gag protein p6 / Non-ribosomal Peptide Synthetase Peptidyl Carrier Protein; Chain A / 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 ...Retrovirus capsid C-terminal domain / Human Immunodeficiency Virus Type 1 Capsid Protein / Human Immunodeficiency Virus Type 1 Capsid Protein / Gag protein p6 / Gag protein p6 / Non-ribosomal Peptide Synthetase Peptidyl Carrier Protein; Chain A / 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. / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 3.9 Å
AuthorsSchur, F.K.M. / Obr, M. / Hagen, W.J.H. / Wan, W. / Arjen, J.J. / Kirkpatrick, J.M. / Sachse, C. / Kraeusslich, H.-G. / Briggs, J.A.G.
Funding support Germany, 2items
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, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2016Group: Database references
Revision 1.2Feb 22, 2017Group: Derived calculations
Revision 1.3Aug 2, 2017Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.name / _em_software.version
Revision 1.4Oct 17, 2018Group: Data collection / Other / Refinement description / Category: cell / refine / Item: _cell.Z_PDB / _refine.pdbx_refine_id
Revision 1.5Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein p24
B: Capsid protein p24
C: Capsid protein p24


Theoretical massNumber of molelcules
Total (without water)74,3683
Polymers74,3683
Non-polymers00
Water0
1
A: Capsid protein p24
B: Capsid protein p24
C: Capsid protein p24
x 6


Theoretical massNumber of molelcules
Total (without water)446,20918
Polymers446,20918
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5

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Components

#1: Protein Capsid protein p24 / / Pr55Gag


Mass: 24789.396 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate NY5)
Strain: isolate NY5 / Gene: gag / Production host: Escherichia coli (E. coli) / References: UniProt: P12493

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Human immunodeficiency virus 1Subtypes of HIV / Type: VIRUS
Details: Virus-like particles were obtained by in vitro assembly of a truncated Gag construct (deltaMACANCSP2) in presence of the maturation inhibitor Bevirimat
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET11C
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 8
Details: Virus-like particles were assembled in the presence of nucleic acid (73mer oligonucleotide, 1:10 molar ratio oligonucleotide:protein).
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2100 mMSodium ChlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acid1
41 mMTCEP1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Virus-like particles were assembled in vitro
Specimen supportDetails: at 20 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 15 K
Details: 10nM colloidal gold was added to the sample prior to plunge freezing.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Nanoprobe
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 3.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2
Details: Number of frames ranged from 8-10 Exposure time per tilt ranged from 0.8 to 1.0 seconds
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 10 / Used frames/image: 8-10

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4CTFFIND4CTF correctionCTF determination
5CTFPHASEFLIPCTF correctionCTF correction within IMOD
8UCSF Chimeramodel fittingrigid body docking using Fit in map option
10CCP4 packagemodel refinement
11PHENIXmodel refinement
12Coot0.8.1model refinement
14AV3final Euler assignment
15TOM Toolboxfinal Euler assignment
17AV33D reconstruction
18TOM Toolbox3D reconstruction
Image processingDetails: Frames were aligned using MotionCorr. Tilts in a tilt series were exposure filtered for cumulative electron dose. Tomograms were reconstructed using IMOD.
CTF correctionDetails: CTF correction was performed using the ctfphaseflip program in IMOD prior to backprojection.
Type: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128733 / Num. of class averages: 1 / Symmetry type: POINT
EM volume selectionDetails: Subtomograms were extracted from the surface of each particle according to the determined radius of the particle.
Num. of tomograms: 43 / Num. of volumes extracted: 527528
Atomic model buildingProtocol: OTHER
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
11L6N

1l6n

11148-279
23DS2

3ds2

11280-353
31U57

1u57

21354-371
RefinementHighest resolution: 3.9 Å

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