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- PDB-9bh6: Human DNA polymerase theta helicase domain tetramer in the apo form -

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Basic information

Entry
Database: PDB / ID: 9bh6
TitleHuman DNA polymerase theta helicase domain tetramer in the apo form
ComponentsDNA polymerase thetaPOLQ
KeywordsDNA BINDING PROTEIN / DNA repair / TMEJ / MMEJ
Function / homology
Function and homology information


single-stranded DNA endodeoxyribonuclease activity / double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / negative regulation of double-strand break repair via homologous recombination / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis ...single-stranded DNA endodeoxyribonuclease activity / double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / negative regulation of double-strand break repair via homologous recombination / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / 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
DNA polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain ...DNA polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZerio, C.J. / Lander, G.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)F32CA288144 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD032467 United States
CitationJournal: 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
DepositionApr 19, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase theta
B: DNA polymerase theta
C: DNA polymerase theta
D: DNA polymerase theta


Theoretical massNumber of molelcules
Total (without water)398,6854
Polymers398,6854
Non-polymers00
Water0
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
DNA polymerase theta / POLQ / DNA polymerase eta


Mass: 99671.344 Da / Num. of mol.: 4
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

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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 DNA polymerase theta helicase domain / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta(DE3) / Plasmid: pET-Duet-1
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepes-NaOH1
2250 mMSodium chlorideNaClSodium chloride1
30.5 mMTCEP1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
VitrificationInstrument: 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 with Whatman 1 filter paper until 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid ...Details: 3 microliters of sample was applied to the surface of the grid, blotted with Whatman 1 filter paper until 2 seconds after the liquid spot on the filter paper stopped spreading, and the grid was plunged into a liquid ethane pool cooled by liquid nitrogen using a manual plunge freezer.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 150 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K
Image recordingAverage exposure time: 0.9 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: 3906
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3particle selectionTemplate picker
2Leginon3.6image acquisition
4cryoSPARC4.3CTF correctionLive Patch CTF
7UCSF ChimeraX1.6.1model fitting
8ISOLDE1.6.0model fittingflexible local modeling
10PHENIX1.20.1model refinementrigid real-space refinement
11cryoSPARC4.3initial Euler assignment
12cryoSPARC4.3final Euler assignment
13cryoSPARC4.3classification
14cryoSPARC4.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1474207
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208545 / Algorithm: FOURIER SPACE
Details: We performed local refinement of a single protomer and then multiplied the output protomer map to fit the D2 symmetric tetramer map.
Symmetry type: POINT
Atomic model buildingB value: 54.43 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: We used ISOLDE to flexibly fit one protomer into the locally refined protomer map. The output protomer model was multiplied and we used ISOLDE to fit four protomers into the tetramer map.
Atomic model buildingPDB-ID: 5A9J

5a9j


Pdb chain-ID: A / Accession code: 5A9J / Chain residue range: 67-892 / Pdb chain residue range: 67-892 / Source name: PDB / Type: experimental model

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