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Yorodumi- EMDB-13139: Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptam... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13139 | |||||||||
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Title | Cryo-EM structure of HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment 166 at the transient P-pocket | |||||||||
Map data | ||||||||||
Sample |
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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 | |||||||||
Biological species | Human immunodeficiency virus type 1 BH10 / Human immunodeficiency virus type 1 group M subtype B (isolate BH10) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.38 Å | |||||||||
Authors | Singh AK / Das K | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13139.map.gz | 6.9 MB | EMDB map data format | |
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Header (meta data) | emd-13139-v30.xml emd-13139.xml | 22.7 KB 22.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13139_fsc.xml | 6.9 KB | Display | FSC data file |
Images | emd_13139.png | 180.5 KB | ||
Others | emd_13139_half_map_1.map.gz emd_13139_half_map_2.map.gz | 6.9 MB 6.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13139 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13139 | HTTPS FTP |
-Related structure data
Related structure data | 7ozwMC 7oxqC 7oz2C 7oz5C 7p15C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_13139.map.gz / Format: CCP4 / Size: 7.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.97 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: #2
File | emd_13139_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13139_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : HIV-1 reverse transcriptase with a DNA aptamer in complex with fr...
Entire | Name: HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment 166 at the transient P-pocket |
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Components |
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-Supramolecule #1: HIV-1 reverse transcriptase with a DNA aptamer in complex with fr...
Supramolecule | Name: HIV-1 reverse transcriptase with a DNA aptamer in complex with fragment 166 at the transient P-pocket type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Human immunodeficiency virus type 1 BH10 |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Molecular weight | Theoretical: 128.4 KDa |
-Supramolecule #2: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT
Supramolecule | Name: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Human immunodeficiency virus type 1 BH10 |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Supramolecule #3: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT
Supramolecule | Name: HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: Human immunodeficiency virus type 1 BH10 |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Macromolecule #1: Reverse transcriptase/ribonuclease H
Macromolecule | Name: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase |
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Source (natural) | Organism: Human immunodeficiency virus type 1 group M subtype B (isolate BH10) Strain: isolate BH10 |
Molecular weight | Theoretical: 64.047398 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ...String: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ILEPFKKQNP DIVIYQYMDD LYVGSDLEIG QHRTKIEELR QHLLRWGLTT PDKKHQKEPP FLWMGYELHP DK WTVQPIV LPEKDSWTVN DIQKLVGKLN WASQIYPGIK VRQLSKLLRG TKALTEVIPL TEEAELELAE NREILKEPVH GVY YDPSKD LIAEIQKQGQ GQWTYQIYQE PFKNLKTGKY ARMRGAHTND VKQLTEAVQK ITTESIVIWG KTPKFKLPIQ KETW ETWWT EYWQATWIPE WEFVNTPPLV KLWYQLEKEP IVGAETFYVD GAANRETKLG KAGYVTNKGR QKVVPLTNTT NQKTE LQAI YLALQDSGLE VNIVTNSQYA LGIIQAQPDK SESELVNQII EQLIKKEKVY LAWVPAHKGI GGNEQVDKLV SA |
-Macromolecule #2: Reverse transcriptase/ribonuclease H
Macromolecule | Name: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase |
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Source (natural) | Organism: Human immunodeficiency virus type 1 BH10 |
Molecular weight | Theoretical: 50.039488 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNET PGIRYQYNVL PQGWKGSPAI F QSSMTKIL ...String: PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWE VQLGIPHPAG LKKKKSVTVL DVGDAYFSVP LDEDFRKYTA FTIPSINNET PGIRYQYNVL PQGWKGSPAI F QSSMTKIL EPFKKQNPDI VIYQYMDDLY VGSDLEIGQH RTKIEELRQH LLRWGLTTPD KKHQKEPPFL WMGYELHPDK WT VQPIVLP EKDSWTVNDI QKLVGKLNWA SQIYPGIKVR QLSKLLRGTK ALTEVIPLTE EAELELAENR EILKEPVHGV YYD PSKDLI AEIQKQGQGQ WTYQIYQEPF KNLKTGKYAR MRGAHTNDVK QLTEAVQKIT TESIVIWGKT PKFKLPIQKE TWET WWTEY WQATWIPEWE FVNTPPLVKL WYQ |
-Macromolecule #3: DNA (37-MER)
Macromolecule | Name: DNA (37-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 11.434332 KDa |
Sequence | String: (DT)(DA)(DA)(DT)(DA)(DT)(OMC)(DC)(OMC)(DC) (DC)(DC)(DT)(DT)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DT)(DT)(DG)(DC)(DA)(DC)(DC)(DG) (DA)(DA)(DG)(DG)(DG)(DG)(DG)(DG) |
-Macromolecule #4: (1~{R},2~{R})-2-phenyl-~{N}-(1,3-thiazol-2-yl)cyclopropane-1-carb...
Macromolecule | Name: (1~{R},2~{R})-2-phenyl-~{N}-(1,3-thiazol-2-yl)cyclopropane-1-carboxamide type: ligand / ID: 4 / Number of copies: 1 / Formula: 3IR |
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Molecular weight | Theoretical: 244.312 Da |
Chemical component information | ChemComp-3IR: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.4 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281 K / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | TFS GLACIOS |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 660 / Average exposure time: 55.0 sec. / Average electron dose: 50.0 e/Å2 |