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- PDB-9mxr: Cryo-EM Structure of HIV-1 Reverse Transcriptase p66 Homodimer in... -

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Basic information

Entry
Database: PDB / ID: 9mxr
TitleCryo-EM Structure of HIV-1 Reverse Transcriptase p66 Homodimer in Complex with 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy] phenoxy}naphthalene-2-carbonitrile (JLJ648), a Non-nucleoside Inhibitor
ComponentsReverse transcriptase/ribonuclease H
KeywordsVIRAL PROTEIN / REVERSE TRANSCRIPTASE / ANTIVIRAL / DRUG DESIGN / HIV-1
Function / homology
Function and homology information


HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency ...HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA stem-loop binding / 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 / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / 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 / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Retrovirus capsid, C-terminal / 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
: / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsHollander, K. / Devarkar, S.C. / Tang, S. / Ma, S. / Xiong, Y. / Jorgensen, W.L. / Anderson, K.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH/NAD AI155072 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI186613 United States
CitationJournal: NPJ Drug Discov / Year: 2025
Title: Mechanistic basis for a novel dual-function Gag-Pol dimerizer potentiating CARD8 inflammasome activation and clearance of HIV-infected cells.
Authors: Klarissa Hollander / Swapnil C Devarkar / Su Tang / Ritudhwaj Tiwari / Shumeng Ma / Won Gil Lee / Elizabeth Denn / Qiankun Wang / Krasimir A Spasov / Jake A Robbins / Kathleen M Frey / ...Authors: Klarissa Hollander / Swapnil C Devarkar / Su Tang / Ritudhwaj Tiwari / Shumeng Ma / Won Gil Lee / Elizabeth Denn / Qiankun Wang / Krasimir A Spasov / Jake A Robbins / Kathleen M Frey / William L Jorgensen / Yong Xiong / Liang Shan / Karen S Anderson /
Abstract: A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside reverse transcriptase inhibitors (NNRTIs) that promote pyroptosis of HIV-1 infected cells. These ...A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside reverse transcriptase inhibitors (NNRTIs) that promote pyroptosis of HIV-1 infected cells. These NNRTIs stimulate dimerization of the Gag-Pol polyprotein, resulting in premature HIV-1 protease (PR) dimerization and cleavage of intracellular CARD8. A unique cell-based high-throughput screen was developed to identify potent compounds activating the CARD8 inflammasome through Gag-Pol dimerization. Our in-house library of NNRTIs was evaluated, including a series of catechol diethers, which are potent, nontoxic antivirals. JLJ648 was identified as a promising dual-function antiviral and Gag-Pol dimerizer. Cryo-EM studies of HIV reverse transcriptase p66 bound to JLJ648 revealed populations of homodimers and, surprisingly, a homotetramer. This novel homotetramer structure resembling an 'infinity knot' revealed two JLJ648-bound homodimers forming an extensive interface and nucleated around a dimer of JLJ648 molecules. Structure-guided mutagenesis studies indicate that Gag-Pol homotetramerization may play a critical role in facilitating PR self-cleavage and triggering pyroptosis.
History
DepositionJan 20, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 17, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Reverse transcriptase/ribonuclease H
B: Reverse transcriptase/ribonuclease H
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,2483
Polymers129,8332
Non-polymers4151
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Reverse transcriptase/ribonuclease H / Exoribonuclease H / p66 RT


Mass: 64916.348 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H
#2: Chemical ChemComp-A1BTU / 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy]phenoxy}naphthalene-2-carbonitrile


Mass: 415.464 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H17N3O3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Human immunodeficiency virus 1 / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 7 / Details: 50mM Tris pH 7.0, 25mM NaCl, 5% Glycerol, 1mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
225 mMSodium ChlorideNaCl1
31 mMTCEPC9H15O6P1
45 PercentGlycerolC3H8O31
SpecimenConc.: 0.65 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
12cryoSPARC3D reconstruction
13PHENIX1.21.2_5419:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 316118 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4WE1

4we1


Accession code: 4WE1 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.05 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037658
ELECTRON MICROSCOPYf_angle_d0.54410417
ELECTRON MICROSCOPYf_dihedral_angle_d13.3042851
ELECTRON MICROSCOPYf_chiral_restr0.0421138
ELECTRON MICROSCOPYf_plane_restr0.0051307

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