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- EMDB-44534: Human DNA polymerase theta helicase domain tetramer in the apo form -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-44534
TitleHuman DNA polymerase theta helicase domain tetramer in the apo form
Map dataCombined tetramer map
Sample
  • Complex: Human DNA polymerase theta helicase domain
    • Protein or peptide: DNA polymerase thetaPOLQ
KeywordsDNA repair / TMEJ / MMEJ / DNA binding protein
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 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: To Be Published
Title: Human polymerase theta helicase positions DNA microhomologies for double-strand break repair
Authors: Zerio CJ / Bai Y / Sosa-Alvarado BA / Guzi T / Lander GC
History
DepositionApr 19, 2024-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44534.png

Downloads & links

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Map

FileDownload / File: emd_44534.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCombined tetramer map
Voxel sizeX=Y=Z: 0.833 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.20019908 - 5.2713566
Average (Standard dev.)0.0051570022 (±0.046421453)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44534_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally refined protomer map

Fileemd_44534_additional_1.map
AnnotationLocally refined protomer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: D2 symmetric tetramer map

Fileemd_44534_additional_2.map
AnnotationD2 symmetric tetramer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Protomer half map A

Fileemd_44534_half_map_1.map
AnnotationProtomer half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Protomer half map B

Fileemd_44534_half_map_2.map
AnnotationProtomer half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human DNA polymerase theta helicase domain

EntireName: Human DNA polymerase theta helicase domain
Components
  • Complex: Human DNA polymerase theta helicase domain
    • Protein or peptide: DNA polymerase thetaPOLQ

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Supramolecule #1: Human DNA polymerase theta helicase domain

SupramoleculeName: Human DNA polymerase theta helicase domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 4 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
10.0 mMHepes-NaOH
250.0 mMSodium chlorideNaClSodium chloride
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 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 60024 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
TemperatureMin: 70.0 K / Max: 77.0 K
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: 3906 / Average exposure time: 0.9 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1474207
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5a9j

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3) / Details: Ab-initio
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3) / Details: Local refinement
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3)
Details: We performed local refinement of a single protomer and then multiplied the output protomer map to fit the D2 symmetric tetramer map.
Number images used: 208545
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5a9j


Chain - Chain ID: A / Chain - Residue range: 67-892 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsWe 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 54.43 / Target criteria: Cross-correlation coefficient
Output model

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

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