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- PDB-7p15: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7p15 | ||||||
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Title | Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment F04 at the transient P-pocket | ||||||
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![]() | TRANSFERASE / Reverse transcriptase / RT-aptamer complex / RT sliding / P-1 complex / P51 / P66 | ||||||
Function / homology | ![]() HIV-1 retropepsin / 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 ...HIV-1 retropepsin / 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 / host cell / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / symbiont-mediated suppression of host gene expression / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / proteolysis / DNA binding / zinc ion binding / membrane Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å | ||||||
![]() | Singh, A.K. / Das, K. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 205.8 KB | Display | ![]() |
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PDB format | ![]() | 157.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.3 MB | Display | ![]() |
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Full document | ![]() | 1.4 MB | Display | |
Data in XML | ![]() | 38.8 KB | Display | |
Data in CIF | ![]() | 58.3 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 13156MC ![]() 7oxqC ![]() 7oz2C ![]() 7oz5C ![]() 7ozwC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 64047.398 Da / Num. of mol.: 1 / Fragment: P66 subunit Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: gag-pol / Plasmid: PCDF-2 EK/LIC / Production host: ![]() ![]() References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H |
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#2: Protein | Mass: 50039.488 Da / Num. of mol.: 1 / Fragment: P51 subunit Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: gag-pol / Plasmid: PCDF-2 EK/LIC / Production host: ![]() ![]() References: UniProt: P03366, RNA-directed DNA polymerase, DNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H |
#3: DNA chain | Mass: 11434.332 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: Chemical | ChemComp-4OI / ( |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Buffer solution | pH: 8 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281 K |
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Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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: 767 |
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Processing
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 733223 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157094 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 148.9 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5HP1 Accession code: 5HP1 / Source name: PDB / Type: experimental model |