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- EMDB-28075: Structure of Lates calcarifer DNA polymerase theta polymerase dom... -

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Basic information

Entry
Database: EMDB / ID: EMD-28075
TitleStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Map dataStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Sample
  • Complex: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide
    • Protein or peptide: DNA polymerase thetaPOLQ
    • DNA: DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')
    • DNA: DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Biological speciesLates calcarifer (barramundi perch)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsLi C / Zhu H / Sun J / Gao Y
Funding support United States, 2 items
OrganizationGrant numberCountry
American Cancer SocietyRSG-22-082-01-DMC United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR170062 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural basis of DNA polymerase θ mediated DNA end joining.
Authors: Chuxuan Li / Hanwen Zhu / Shikai Jin / Leora M Maksoud / Nikhil Jain / Ji Sun / Yang Gao /
Abstract: DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes ...DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.
History
DepositionSep 8, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28075.png

Downloads & links

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Map

FileDownload / File: emd_28075.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 1.3
Minimum - Maximum-9.238321 - 15.36017
Average (Standard dev.)-0.0011530876 (±0.16275579)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Structure of Lates calcarifer DNA polymerase theta polymerase...

Fileemd_28075_half_map_1.map
AnnotationStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of Lates calcarifer DNA polymerase theta polymerase...

Fileemd_28075_half_map_2.map
AnnotationStructure of Lates calcarifer DNA polymerase theta polymerase domain with long duplex DNA, complex Ia
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Lates calcarifer DNA polymerase Theta with dup...

EntireName: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide
Components
  • Complex: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide
    • Protein or peptide: DNA polymerase thetaPOLQ
    • DNA: DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')
    • DNA: DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of Lates calcarifer DNA polymerase Theta with dup...

SupramoleculeName: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Lates calcarifer (barramundi perch)

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lates calcarifer (barramundi perch)
Molecular weightTheoretical: 95.674117 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGHHHHHHPS GVKTENNDHI NLKVAGQDGS VVQFKIKRHT PLSKLMKAYC ERQGLSMRQI RFRFDGQPIN ETDTPAQLEM EDEDTIDVF QQQTGGCMDP PSDAESPVTD DGFTLQLSQD ASLCPSNSGT FSIIDVASDR RLFNTFIKEW KTKERYSLAL A CEKREHIQ ...String:
MGHHHHHHPS GVKTENNDHI NLKVAGQDGS VVQFKIKRHT PLSKLMKAYC ERQGLSMRQI RFRFDGQPIN ETDTPAQLEM EDEDTIDVF QQQTGGCMDP PSDAESPVTD DGFTLQLSQD ASLCPSNSGT FSIIDVASDR RLFNTFIKEW KTKERYSLAL A CEKREHIQ QPEGEIGGKH KRAPAARQKL NRTDGFPVRD SDGLVLIGLS VCWGARDSYY ISLQQEQSKG LSSSLAPPPL DD DLPVSER LGQVRSCLSR PSAGLRGGVV VTYDIIQVYK TLVLSCGISL AGNCEDPKVA CWLLDPGSEE RTLPNMVTVY CPE ELPLLD GLGSAHAHCP RVRAATKSVL VHAVMNHLTG LLEKDSMLDL FRSIEMPSQV CLALLELNGV GFSVEECERQ KHVM QAKLT ALESQAYNLA GHSFSLTSID DIAQVLFLEL HLPPNGDVGG SKSKKTLGYT RRGGGRVRLG KQFSTTKDIL EKLRP LHPL PGVILEWRRI TNALTKVVFP LQREKQYHPT LAMDRIYPIA QTHTATGRVS FTEPNIQNVP KDFEICMPTV VGESPP SQN GCQMTTKPGK NRRSVAPSVT GGAAEQGPAF SVSMRHAFVP FSGGMILAAD YSQLELRVLA HLSKDQRLLQ VLNGGAD VF RCIAAEWKGV DPETVNDSLR QQAKQICYGI IYGMGAKSLG EQMGVEENDA ACYIESFKAR YKGINAFLKE TVKNCIKN G YVQTLMGRRR YLPGISNTNT HIKAHAERQA VNTTVQGSAA DIVKLATVNI QKRLRKTYPT APLSHQHTHS GTSQYRAGT SHLRGAFFVL QLHDELIYET REEDLIQVAQ IVKREMESAV KLYVKLKAKV KVGPSWGNLQ DLDL

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Macromolecule #2: DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')

MacromoleculeName: DNA (5'-D(*AP*GP*CP*TP*CP*TP*AP*CP*GP*GP*AP*TP*GP*C)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lates calcarifer (barramundi perch)
Molecular weightTheoretical: 8.864712 KDa
SequenceString:
(DG)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DC)(DT) (DC)(DT)(DA)(DC)(DG)(DG)(DA)(DT)(DG)(DC) (DC)(DT)(DC)(DA)(DC)(DA)(DG)(DC)(DA)

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Macromolecule #3: DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')

MacromoleculeName: DNA (5'-D(*AP*GP*CP*AP*TP*CP*CP*GP*TP*AP*GP*(2DA))-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lates calcarifer (barramundi perch)
Molecular weightTheoretical: 5.844796 KDa
SequenceString:
(DT)(DG)(DC)(DT)(DG)(DT)(DG)(DA)(DG)(DC) (DA)(DT)(DC)(DC)(DG)(DT)(DA)(DG)(2DA)

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Macromolecule #4: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: DGT
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-DGT:
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / Deoxyguanosine triphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.6
GridModel: UltrAuFoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 825204

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