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- PDB-7sjx: Cryo-EM Structure of the PR-RT components of the HIV-1 Pol Polyprotein -

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Basic information

Entry
Database: PDB / ID: 7sjx
TitleCryo-EM Structure of the PR-RT components of the HIV-1 Pol Polyprotein
ComponentsGag-Pol polyprotein
KeywordsVIRAL PROTEIN / HIV-1 / Reverse transcriptase / Protease / enzyme
Function / homology
Function and homology information


HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding ...HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / viral penetration into host nucleus / host multivesicular body / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont-mediated suppression of host gene expression / viral translational frameshifting / lipid binding / symbiont entry into host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / zinc ion binding / membrane
Similarity search - Function
Reverse transcriptase connection / Reverse transcriptase connection domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain ...Reverse transcriptase connection / Reverse transcriptase connection domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / RNase H type-1 domain profile. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsLyumkis, D. / Passos, D. / Arnold, E. / Harrison, J.J.E.K. / Ruiz, F.X.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI136680 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI27690 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54AI150472 United States
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-EM structure of the HIV-1 Pol polyprotein provides insights into virion maturation.
Authors: Jerry Joe E K Harrison / Dario Oliveira Passos / Jessica F Bruhn / Joseph D Bauman / Lynda Tuberty / Jeffrey J DeStefano / Francesc Xavier Ruiz / Dmitry Lyumkis / Eddy Arnold /
Abstract: Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields ...Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo-electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.
History
DepositionOct 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 27, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Aug 16, 2023Group: Data collection / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_database_status / pdbx_initial_refinement_model
Item: _pdbx_database_status.pdb_format_compatible

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gag-Pol polyprotein
B: Gag-Pol polyprotein


Theoretical massNumber of molelcules
Total (without water)238,5802
Polymers238,5802
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Gag-Pol polyprotein / Pr160Gag-Pol


Mass: 119289.867 Da / Num. of mol.: 2 / Fragment: PR-RT portion, residues 479-1447 / Mutation: D131A, L765D, F766D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Strain: isolate BH10 / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P03366

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PR-RT portion of HIV-1 Pol / Type: COMPLEX
Details: 3D reconstruction of the HIV-1 Pol polyprotein comprising the PR-RT portion
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Human immunodeficiency virus type 1 BH10
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Image recordingElectron dose: 0.95 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 60

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1Warpparticle selection
4WarpCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11RosettaEMmodel refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cisTEM1classification
15cisTEM13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 27555
3D reconstructionResolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27555 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coeficient
Atomic model building
IDPDB-ID 3D fitting-ID
11DLO

1dlo

1
23BGR

3bgr

1
32HB4

2hb4

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 526.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00759451
ELECTRON MICROSCOPYf_angle_d0.92812836
ELECTRON MICROSCOPYf_chiral_restr0.06511419
ELECTRON MICROSCOPYf_plane_restr0.00641610
ELECTRON MICROSCOPYf_dihedral_angle_d14.48513577

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