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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 thetaPOLQ
    • 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


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) / 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: 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_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
Voxel sizeX=Y=Z: 0.833 Å
Density
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
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharp map

Fileemd_44537_additional_1.map
AnnotationSharp map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_44537_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_44537_half_map_2.map
AnnotationHalf 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 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 thetaPOLQ
    • 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 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 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
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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
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: 12440 / Average exposure time: 1.4 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: 5424691
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5a9j


Details: 2P6R was also used for DNA
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: Non-uniform refinement
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
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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