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Yorodumi- PDB-9bh9: Human DNA polymerase theta helicase domain dimer bound to DNA in ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 9bh9 | |||||||||
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Title | Human DNA polymerase theta helicase domain dimer bound to DNA in the microhomology aligning conformation | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN / DNA repair / TMEJ / MMEJ | |||||||||
Function / homology | Function and homology information single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis ...single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis / DNA helicase activity / base-excision repair / protein homooligomerization / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / double-strand break repair / site of double-strand break / DNA helicase / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / DNA damage response / chromatin binding / Golgi apparatus / magnesium ion binding / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / cytosol Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Zerio, C.J. / Lander, G.C. | |||||||||
Funding support | United States, 2items
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Citation | Journal: To Be Published Title: Human polymerase theta helicase positions DNA microhomologies for double-strand break repair Authors: Zerio, C.J. / Bai, Y. / Sosa-Alvarado, B.A. / Guzi, T. / Lander, G.C. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 9bh9.cif.gz | 302.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb9bh9.ent.gz | 237.7 KB | Display | PDB format |
PDBx/mmJSON format | 9bh9.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 9bh9_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 9bh9_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 9bh9_validation.xml.gz | 54.8 KB | Display | |
Data in CIF | 9bh9_validation.cif.gz | 82.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bh/9bh9 ftp://data.pdbj.org/pub/pdb/validation_reports/bh/9bh9 | HTTPS FTP |
-Related structure data
Related structure data | 44537MC 9bh6C 9bh7C 9bh8C 9bhaC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 99671.344 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLQ, POLH / Plasmid: pET-Duet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta References: UniProt: O75417, DNA helicase, DNA-directed DNA polymerase, RNA-directed DNA polymerase #2: DNA chain | Mass: 17264.012 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight |
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: We added 2X pre-annealed DNA to the protein, lowered the NaCl concentration to 100 mM, and purified the DNA-bound complex by SEC. | ||||||||||||||||||||||||
Specimen support | Details: Grids were glow discharged under vacuum for 30 s at 15 mA in a Pelco easiGlow 91000 Glow Discharge Cleaning System. Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K Details: 3 microliters of sample was applied to the surface of the grid, blotted for 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ...Details: 3 microliters of sample was applied to the surface of the grid, blotted for 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ethane bath cooled by liquid nitrogen. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 60024 X / Nominal defocus max: 1000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 150 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K |
Image recording | Average exposure time: 1.4 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12440 |
EM imaging optics | Energyfilter slit width: 20 eV |
Image scans | Sampling size: 5 µm / Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 5424691 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107353 / Algorithm: FOURIER SPACE Details: Non-uniform refinement and local filtering by resolution Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 67.19 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient Details: We modeled DNA bases into sharpened density when possible. We modeled the DNA backbone into locally filtered density in low resolution areas. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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