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- PDB-7ozw: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam... -

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Basic information

Entry
Database: PDB / ID: 7ozw
TitleCryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment 166 at the transient P-pocket
Components
  • (Reverse transcriptase/ribonuclease H) x 2
  • DNA (37-MER)
KeywordsTRANSFERASE / Reverse transcriptase / RT-aptamer complex / RT sliding / P-1 complex / P51 / P66
Function / homology
Function and homology information


HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA ...HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / viral penetration into host nucleus / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / lipid binding / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / RNA 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. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / 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. / Ribonuclease H superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-3IR / DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
Human immunodeficiency virus type 1 BH10
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsSingh, A.K. / Das, K.
CitationJournal: Nat Commun / Year: 2021
Title: Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.
Authors: Abhimanyu K Singh / Sergio E Martinez / Weijie Gu / Hoai Nguyen / Dominique Schols / Piet Herdewijn / Steven De Jonghe / Kalyan Das /
Abstract: HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the ...HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.
History
DepositionJun 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 22, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Reverse transcriptase/ribonuclease H
B: Reverse transcriptase/ribonuclease H
F: DNA (37-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,7664
Polymers125,5213
Non-polymers2441
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10340 Å2
ΔGint-39 kcal/mol
Surface area47330 Å2
MethodPISA

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Components

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


Mass: 64047.398 Da / Num. of mol.: 1 / Fragment: P66 subunit
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 / Plasmid: PCDF-2 EK/LIC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21 codon plus RIL
References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H
#2: Protein Reverse transcriptase/ribonuclease H / Exoribonuclease H / p51 RT


Mass: 50039.488 Da / Num. of mol.: 1 / Fragment: P51 subunit
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 BH10
Gene: gag-pol / Plasmid: PCDF-2 EK/LIC / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21 codon plus RIL
References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H
#3: DNA chain DNA (37-MER)


Mass: 11434.332 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-3IR / (1~{R},2~{R})-2-phenyl-~{N}-(1,3-thiazol-2-yl)cyclopropane-1-carboxamide / ((1R,2R)-2-Phenyl-N-(1,3-thiazol-2-yl) cyclopropanecarboxamide


Mass: 244.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H12N2OS / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment 166 at the transient P-pocketCOMPLEX#1-#30RECOMBINANT
2HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNITCOMPLEX#11RECOMBINANT
3HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNITCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.1284 MDaNO
12
13
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Human immunodeficiency virus type 1 BH1011678
32Human immunodeficiency virus type 1 BH1011678
43Human immunodeficiency virus type 1 BH1011678
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-HClTrisNH2C(CH2OH)3HCl1
275 mMNaClSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.4 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: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 150000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 55 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 660

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.9.0image acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 856171
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 146670 / Symmetry type: POINT
Atomic model buildingB value: 140.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5HP1

5hp1

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038701
ELECTRON MICROSCOPYf_angle_d0.54211991
ELECTRON MICROSCOPYf_dihedral_angle_d15.0951352
ELECTRON MICROSCOPYf_chiral_restr0.0411320
ELECTRON MICROSCOPYf_plane_restr0.0051395

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