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- EMDB-44537: Human DNA polymerase theta helicase domain dimer bound to DNA in ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44537
TitleHuman DNA polymerase theta helicase domain dimer bound to DNA in the microhomology aligning conformation
Map dataMap locally filtered by resolution
Sample
  • Complex: Human DNA polymerase theta helicase domain bound to stem-loop DNA with microhomology in the 3' overhang
    • Complex: Human DNA polymerase theta helicase domain
      • Protein or peptide: DNA polymerase theta
    • Complex: Stem-loop DNA with microhomology in the 3' overhang
      • DNA: Stem-loop DNA with microhomology in the 3' overhang
KeywordsDNA repair / TMEJ / MMEJ / DNA binding protein
Function / homology
Function and homology information


double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / replication fork processing / site of DNA damage / mitochondrial nucleoid / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination ...double-strand break repair via alternative nonhomologous end joining / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / replication fork processing / site of DNA damage / mitochondrial nucleoid / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / error-prone translesion synthesis / negative regulation of double-strand break repair via homologous recombination / RNA-directed DNA polymerase activity / DNA helicase activity / base-excision repair / protein homooligomerization / RNA-directed DNA polymerase / 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 / magnesium ion binding / Golgi apparatus / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
: / DNA_pol_Q helicase like region helical domain / DNA polymerase theta-like, helix-turn-helix domain / Helix-turn-helix 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_pol_Q helicase like region helical domain / DNA polymerase theta-like, helix-turn-helix domain / Helix-turn-helix 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 domain / DEAD/DEAH box helicase / 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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZerio CJ / Lander GC
Funding support United States, 2 items
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: Nat Struct Mol Biol / Year: 2025
Title: Human polymerase θ helicase positions DNA microhomologies for double-strand break repair.
Authors: Christopher J Zerio / Yonghong Bai / Brian A Sosa-Alvarado / Timothy Guzi / Gabriel C Lander /
Abstract: DNA double-strand breaks occur daily in all human cells and must be repaired with high fidelity to minimize genomic instability. Deficiencies in high-fidelity DNA repair by homologous recombination ...DNA double-strand breaks occur daily in all human cells and must be repaired with high fidelity to minimize genomic instability. Deficiencies in high-fidelity DNA repair by homologous recombination lead to dependence on DNA polymerase θ, which identifies DNA microhomologies in 3' single-stranded DNA overhangs and anneals them to initiate error-prone double-strand break repair. The resulting genomic instability is associated with numerous cancers, thereby making this polymerase an attractive therapeutic target. However, despite the biomedical importance of polymerase θ, the molecular details of how it initiates DNA break repair remain unclear. Here, we present cryo-electron microscopy structures of the polymerase θ helicase domain bound to microhomology-containing DNA, revealing DNA-induced rearrangements of the helicase that enable DNA repair. Our structures show that DNA-bound helicase dimers facilitate a microhomology search that positions 3' single-stranded DNA ends in proximity to align complementary bases and anneal DNA microhomology. We characterize the molecular determinants that enable the helicase domain of polymerase θ to identify and pair DNA microhomologies to initiate mutagenic DNA repair, thereby providing insight into potentially targetable interactions for therapeutic interventions.
History
DepositionApr 19, 2024-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44537.png

Downloads & links

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Map

FileDownload / File: emd_44537.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap locally filtered by resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 324 pix.
= 269.892 Å		0.83 Å/pix.
x 324 pix.
= 269.892 Å		0.83 Å/pix.
x 324 pix.
= 269.892 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.3843435 - 2.18983
Average (Standard dev.)0.0023942278 (±0.0397503)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 269.892 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44537_msk_1.map
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Additional map: Sharp map

Fileemd_44537_additional_1.map
AnnotationSharp map
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Half map: Half map A

Fileemd_44537_half_map_1.map
AnnotationHalf map A
Projections & Slices
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Half map: Half map B

Fileemd_44537_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Human DNA polymerase theta helicase domain bound to stem-loop DNA...

EntireName: Human DNA polymerase theta helicase domain bound to stem-loop DNA with microhomology in the 3' overhang
Components
  • Complex: Human DNA polymerase theta helicase domain bound to stem-loop DNA with microhomology in the 3' overhang
    • Complex: Human DNA polymerase theta helicase domain
      • Protein or peptide: DNA polymerase theta
    • Complex: Stem-loop DNA with microhomology in the 3' overhang
      • DNA: Stem-loop DNA with microhomology in the 3' overhang

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Supramolecule #1: Human DNA polymerase theta helicase domain bound to stem-loop DNA...

SupramoleculeName: Human DNA polymerase theta helicase domain bound to stem-loop DNA with microhomology in the 3' overhang
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20 KDa

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Supramolecule #2: Human DNA polymerase theta helicase domain

SupramoleculeName: Human DNA polymerase theta helicase domain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Stem-loop DNA with microhomology in the 3' overhang

SupramoleculeName: Stem-loop DNA with microhomology in the 3' overhang / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.671344 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: NLLRRSGKRR RSESGSDSFS GSGGDSSASP QFLSGSVLSP PPGLGRCLKA AAAGECKPTV PDYERDKLLL ANWGLPKAVL EKYHSFGVK KMFEWQAECL LLGQVLEGKN LVYSAPTSAG KTLVAELLIL KRVLEMRKKA LFILPFVSVA KEKKYYLQSL F QEVGIKVD ...String:
NLLRRSGKRR RSESGSDSFS GSGGDSSASP QFLSGSVLSP PPGLGRCLKA AAAGECKPTV PDYERDKLLL ANWGLPKAVL EKYHSFGVK KMFEWQAECL LLGQVLEGKN LVYSAPTSAG KTLVAELLIL KRVLEMRKKA LFILPFVSVA KEKKYYLQSL F QEVGIKVD GYMGSTSPSR HFSSLDIAVC TIERANGLIN RLIEENKMDL LGMVVVDELH MLGDSHRGYL LELLLTKICY IT RKSASCQ ADLASSLSNA VQIVGMSATL PNLELVASWL NAELYHTDFR PVPLLESVKV GNSIYDSSMK LVREFEPMLQ VKG DEDHVV SLCYETICDN HSVLLFCPSK KWCEKLADII AREFYNLHHQ AEGLVKPSEC PPVILEQKEL LEVMDQLRRL PSGL DSVLQ KTVPWGVAFH HAGLTFEERD IIEGAFRQGL IRVLAATSTL SSGVNLPARR VIIRTPIFGG RPLDILTYKQ MVGRA GRKG VDTVGESILI CKNSEKSKGI ALLQGSLKPV RSCLQRREGE EVTGSMIRAI LEIIVGGVAS TSQDMHTYAA CTFLAA SMK EGKQGIQRNQ ESVQLGAIEA CVMWLLENEF IQSTEASDGT EGKVYHPTHL GSATLSSSLS PADTLDIFAD LQRAMKG FV LENDLHILYL VTPMFEDWTT IDWYRFFCLW EKLPTSMKRV AELVGVEEGF LARCVKGKVV ARTERQHRQM AIHKRFFT S LVLLDLISEV PLREINQKYG CNRGQIQSLQ QSAAVYAGMI TVFSNRLGWH NMELLLSQFQ KRLTFGIQRE LCDLVRVSL LNAQRARVLY ASGFHTVADL ARANIVEVEV ILKNAVPFKS ARKAVDEEEE AVEERRNMRT IWVTGRKGLT EREAAALIVE EARMILQQD LVEM

UniProtKB: DNA polymerase theta

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Macromolecule #2: Stem-loop DNA with microhomology in the 3' overhang

MacromoleculeName: Stem-loop DNA with microhomology in the 3' overhang / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.264012 KDa
SequenceString: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DC)(DA)(DC)(DT) (DG)(DT)(DG)(DA)(DG)(DC)(DT)(DT)(DA) (DG)(DC)(DG)(DT)(DT)(DA)(DG)(DA)(DG)(DT) (DA) (DG)(DG)(DT)(DT)(DT)(DT) ...String:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DC)(DA)(DC)(DT) (DG)(DT)(DG)(DA)(DG)(DC)(DT)(DT)(DA) (DG)(DC)(DG)(DT)(DT)(DA)(DG)(DA)(DG)(DT) (DA) (DG)(DG)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DG)(DC)(DC)(DC)(DG)(DG)(DG)

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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

Concentration1.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHepes-NaOH
100.0 mMSodium chlorideNaCl
0.5 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: Grids were glow discharged under vacuum for 30 s at 15 mA in a Pelco easiGlow 91000 Glow Discharge Cleaning System.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
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..
DetailsWe added 2X pre-annealed DNA to the protein, lowered the NaCl concentration to 100 mM, and purified the DNA-bound complex by SEC.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 12440 / Average exposure time: 1.4 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 60024 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5424691
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5a9j


Details: 2P6R was also used for DNA
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3)
Details: Non-uniform refinement and local filtering by resolution
Number images used: 107353
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3) / Details: Ab-initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3) / Details: Non-uniform refinement
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

5a9j

chain_id: A, residue_range: 67-892, source_name: PDB, initial_model_type: experimental modelProtein

2p6r

chain_id: X, residue_range: 6-19, source_name: PDB, initial_model_type: experimental modelDNA
DetailsWe modeled DNA bases into sharpened density when possible. We modeled the DNA backbone into locally filtered density in low resolution areas.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 67.19 / Target criteria: Cross-correlation coefficient
Output model

PDB-9bh9:
Human DNA polymerase theta helicase domain dimer bound to DNA in the microhomology aligning conformation

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