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- PDB-6n3j: MicroED Structure of the CTD-SP1 fragment of HIV-1 Gag -

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Basic information

Entry
Database: PDB / ID: 6n3j
TitleMicroED Structure of the CTD-SP1 fragment of HIV-1 Gag
ComponentsCTD-SP1 fragment of HIV-1 Gag
KeywordsVIRAL PROTEIN / Bevirimat / HIV-1 Gag / MicroED / Immature Hexagonal Lattice
Function / homology
Function and homology information


host cellular component / Synthesis And Processing Of GAG, GAGPOL Polyproteins / host cell nuclear membrane / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / Uncoating of the HIV Virion / 2-LTR circle formation / Vpr-mediated nuclear import of PICs ...host cellular component / Synthesis And Processing Of GAG, GAGPOL Polyproteins / host cell nuclear membrane / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / Minus-strand DNA synthesis / Plus-strand DNA synthesis / Uncoating of the HIV Virion / 2-LTR circle formation / Vpr-mediated nuclear import of PICs / viral budding via host ESCRT complex / Early Phase of HIV Life Cycle / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / Binding and entry of HIV virion / Membrane binding and targetting of GAG proteins / viral process / Assembly Of The HIV Virion / Budding and maturation of HIV virion / host multivesicular body / viral capsid / viral nucleocapsid / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / membrane
Similarity search - Function
Retrovirus capsid C-terminal domain / 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 domain / 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
Gag protein / Gag polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / molecular replacement / cryo EM / Resolution: 3 Å
Model detailsDrug-free Gag CTD-SP1
AuthorsPurdy, M.D. / Shi, D. / Hattne, J. / Chrustowicz, J.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)5 P50 GM082545-10 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM066087 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P50 GM082545 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM12850 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat.
Authors: Michael D Purdy / Dan Shi / Jakub Chrustowicz / Johan Hattne / Tamir Gonen / Mark Yeager /
Abstract: HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 ...HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.
History
DepositionNov 15, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 12, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Dec 26, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jan 9, 2019Group: Data collection / Database references / Category: citation / diffrn_radiation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _diffrn_radiation.pdbx_diffrn_protocol / _diffrn_radiation.pdbx_scattering_type
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: CTD-SP1 fragment of HIV-1 Gag
B: CTD-SP1 fragment of HIV-1 Gag
C: CTD-SP1 fragment of HIV-1 Gag
D: CTD-SP1 fragment of HIV-1 Gag
E: CTD-SP1 fragment of HIV-1 Gag
F: CTD-SP1 fragment of HIV-1 Gag


Theoretical massNumber of molelcules
Total (without water)73,1576
Polymers73,1576
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7980 Å2
ΔGint-58 kcal/mol
Surface area27320 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.614, 122.552, 78.704
Angle α, β, γ (deg.)90.000, 96.550, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
CTD-SP1 fragment of HIV-1 Gag


Mass: 12192.895 Da / Num. of mol.: 6 / Fragment: CTD-SP1 / Mutation: P373T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: gag / Plasmid: pET30a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: A0A248SMC7, UniProt: P04591*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: CTD-SP1 fragment of HIV-1 Gag / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: .01217387 MDa / Experimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1 / Strain: NL4-3
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pet30b
Details of virusIsolate: STRAIN / Type: VIRION
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 MBis-Tris Propane1
21.1 MLithium sulfateLiSO41
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharge 30s each side / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
CrystalDensity Matthews: 2.31 Å3/Da / Density % sol: 46.81 %
Crystal growTemperature: 295 K / Method: vapor diffusion / pH: 7 / Details: 0.1 M Bis-Tris Propane, 1.1 M LiSO4

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingAverage exposure time: 8 sec. / Electron dose: 0.05 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of grids imaged: 6
Details: Data from 6 crystals was merged for structure determination.
EM diffractionCamera length: 2000 mm
EM diffraction shell
Resolution (Å)IDEM diffraction stats-IDFourier space coverage (%)MultiplicityNum. of structure factorsPhase residual (°)
3-19.91179.85.7104801
3-3.22178.75.710061
EM diffraction statsFourier space coverage: 79.8 % / High resolution: 3 Å / Num. of intensities measured: 10480 / Num. of structure factors: 10480 / Phase error: 0 ° / Phase residual: 1 ° / Phase error rejection criteria: 0 / Rmerge: 0.564 / Rsym: 0.255
Diffraction sourceSource: ELECTRON MICROSCOPE / Wavelength: 0.0251 Å
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.0251 Å / Relative weight: 1
ReflectionResolution: 3→27.5 Å / Num. obs: 10480 / % possible obs: 79.8 % / Redundancy: 5.7 % / Biso Wilson estimate: 55.82 Å2 / CC1/2: 0.9 / Rpim(I) all: 0.255 / Net I/σ(I): 3
Reflection shellResolution: 3→3.2 Å / Redundancy: 5.7 % / Mean I/σ(I) obs: 0.7 / Num. unique all: 1006 / CC1/2: 0.35 / Rpim(I) all: 1.324 / % possible all: 78.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
PHENIXdev_2747refinement
PDB_EXTRACT3.24data extraction
EM software
IDNameVersionCategory
8PHENIXdev-2747molecular replacement
11AIMLESScrystallography merging
13PHENIXdev-2747model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 96.55 ° / A: 70.61 Å / B: 122.55 Å / C: 78.7 Å / Space group name: C121 / Space group num: 5
CTF correctionType: NONE
3D reconstructionResolution: 3 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 41.1 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL
Atomic model buildingPDB-ID: 5I4T

5i4t


Accession code: 5I4T / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5i4t

5i4t


Resolution: 3→19.863 Å / SU ML: 0.59 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 34.73
RfactorNum. reflection% reflection
Rfree0.2916 1049 10.01 %
Rwork0.2536 --
obs0.2576 10477 78.6 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 215.22 Å2 / Biso mean: 46.1189 Å2 / Biso min: 6.76 Å2
Refinement stepCycle: final / Resolution: 3→19.863 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3805 0 0 0 3805
Num. residues----545
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0033883
ELECTRON CRYSTALLOGRAPHYf_angle_d0.5615295
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.034635
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.002686
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d11.3441333
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Rfactor Rfree error: 0 / Total num. of bins used: 7

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.0002-3.15780.44961310.37611286141775
3.1578-3.35470.33931430.33951322146577
3.3547-3.61230.34071610.30621339150079
3.6123-3.97320.31441690.25041364153380
3.9732-4.54210.23441600.20691357151780
4.5421-5.70010.25841250.21471397152280
5.7001-19.86360.25811600.2251363152379

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