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Basic information

Entry
Database: PDB / ID: 6wid
TitleNucleotide incorporation intermediate into quaternary complex of human Polymerase Mu on a complementary DNA double-strand break substrate
Components
  • DNA (5'-D(*AP*CP*G)-3')
  • DNA (5'-D(*CP*GP*GP*CP*AP*T)-3')
  • DNA (5'-D(*CP*GP*TP*AP*T)-3')
  • DNA (5'-D(P*GP*CP*CP*G)-3')
  • DNA-directed DNA/RNA polymerase mu
KeywordsTRANSFERASE/DNA / Family X polymerase / nonhomologous end-joining / DNA double-strand break repair / TRANSFERASE-DNA complex
Function / homology
Function and homology information


Nonhomologous End-Joining (NHEJ) / double-strand break repair via nonhomologous end joining / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
DNA-directed DNA/RNA polymerase mu / DNA nucleotidylexotransferase (TdT) / DNA-directed DNA/RNA polymerase mu / DNA polymerase beta, palm domain / DNA polymerase beta palm / DNA polymerase lambda, fingers domain / Fingers domain of DNA polymerase lambda / DNA-directed DNA polymerase X / DNA polymerase beta-like, N-terminal domain / Helix-hairpin-helix domain / DNA polymerase X family ...DNA-directed DNA/RNA polymerase mu / DNA nucleotidylexotransferase (TdT) / DNA-directed DNA/RNA polymerase mu / DNA polymerase beta, palm domain / DNA polymerase beta palm / DNA polymerase lambda, fingers domain / Fingers domain of DNA polymerase lambda / DNA-directed DNA polymerase X / DNA polymerase beta-like, N-terminal domain / Helix-hairpin-helix domain / DNA polymerase X family / DNA polymerase lambda lyase domain superfamily / DNA polymerase family X, binding site / DNA polymerase family X signature. / DNA polymerase family X / DNA polymerase beta, thumb domain / DNA polymerase beta thumb / DNA polymerase, thumb domain superfamily / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Nucleotidyltransferase superfamily
Similarity search - Domain/homology
2'-DEOXYURIDINE 5'-ALPHA,BETA-IMIDO-TRIPHOSPHATE / DI(HYDROXYETHYL)ETHER / PYROPHOSPHATE / DNA / DNA-directed DNA/RNA polymerase mu
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsKaminski, A.M. / Kunkel, T.A. / Pedersen, L.C. / Bebenek, K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIA ES 102645 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065070 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structural snapshots of human DNA polymerase mu engaged on a DNA double-strand break.
Authors: Kaminski, A.M. / Pryor, J.M. / Ramsden, D.A. / Kunkel, T.A. / Pedersen, L.C. / Bebenek, K.
History
DepositionApr 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed DNA/RNA polymerase mu
T: DNA (5'-D(*CP*GP*GP*CP*AP*T)-3')
U: DNA (5'-D(*AP*CP*G)-3')
P: DNA (5'-D(*CP*GP*TP*AP*T)-3')
D: DNA (5'-D(P*GP*CP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,71815
Polymers45,4375
Non-polymers1,28110
Water6,305350
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7110 Å2
ΔGint-57 kcal/mol
Surface area15700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.186, 62.040, 118.320
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA-directed DNA/RNA polymerase mu / Pol Mu / Terminal transferase


Mass: 40054.434 Da / Num. of mol.: 1 / Fragment: UNP residues 132-494 / Mutation: P398-P410 deletion replaced by G410 linker
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLM, polmu / Plasmid: pGEXM / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Rosetta2 (DE3) / References: UniProt: Q9NP87, DNA-directed DNA polymerase

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DNA chain , 4 types, 4 molecules TUPD

#2: DNA chain DNA (5'-D(*CP*GP*GP*CP*AP*T)-3')


Mass: 1809.217 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*AP*CP*G)-3')


Mass: 886.637 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*CP*GP*TP*AP*T)-3')


Mass: 1495.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain DNA (5'-D(P*GP*CP*CP*G)-3')


Mass: 1191.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 9 types, 360 molecules

#6: Chemical ChemComp-DUP / 2'-DEOXYURIDINE 5'-ALPHA,BETA-IMIDO-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PPV / PYROPHOSPHATE / Pyrophosphate


Mass: 177.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H4O7P2 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#10: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#11: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400 / Polyethylene glycol


Mass: 238.278 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#12: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#13: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#14: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 350 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.43 Å3/Da / Density % sol: 49.4 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: 40-45.5 mM MES, pH 5.6, 0.16-0.182 M potassium chloride, 8.2-9.1 mM magnesium sulfate, 8.2-9.1% w/v PEG400

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Data collection

DiffractionMean temperature: 93 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Dec 5, 2019
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.5→50 Å / Num. obs: 71251 / % possible obs: 99.8 % / Redundancy: 6 % / Biso Wilson estimate: 14.06 Å2 / CC1/2: 0.998 / CC star: 0.999 / Rpim(I) all: 0.037 / Rrim(I) all: 0.091 / Rsym value: 0.083 / Χ2: 1.215 / Net I/σ(I): 28.5
Reflection shellResolution: 1.5→1.53 Å / Redundancy: 5.5 % / Mean I/σ(I) obs: 2.24 / Num. unique obs: 3524 / CC1/2: 0.848 / CC star: 0.958 / Rpim(I) all: 0.255 / Rrim(I) all: 0.606 / Rsym value: 0.548 / Χ2: 0.42 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX1.16_3549refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4M04

4m04


Resolution: 1.5→31.02 Å / SU ML: 0.1371 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 15.8136
RfactorNum. reflection% reflectionSelection details
Rfree0.1774 2195 3.08 %random
Rwork0.1639 ---
obs0.1644 71176 99.64 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 19.95 Å2
Refinement stepCycle: LAST / Resolution: 1.5→31.02 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2562 361 72 350 3345
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00923347
X-RAY DIFFRACTIONf_angle_d1.08724653
X-RAY DIFFRACTIONf_chiral_restr0.0599509
X-RAY DIFFRACTIONf_plane_restr0.0072536
X-RAY DIFFRACTIONf_dihedral_angle_d16.65011253
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.530.22471340.20174151X-RAY DIFFRACTION97.65
1.53-1.570.21351380.17994252X-RAY DIFFRACTION99.93
1.57-1.610.17991380.17044296X-RAY DIFFRACTION99.98
1.61-1.650.18431330.17044262X-RAY DIFFRACTION99.91
1.65-1.70.20611390.15994268X-RAY DIFFRACTION99.71
1.7-1.760.16211330.16364256X-RAY DIFFRACTION99.68
1.76-1.820.19311410.15694279X-RAY DIFFRACTION99.35
1.82-1.890.17761330.1574286X-RAY DIFFRACTION99.77
1.89-1.980.19041370.16134294X-RAY DIFFRACTION99.98
1.98-2.080.17521380.16514320X-RAY DIFFRACTION100
2.08-2.210.18111360.16164303X-RAY DIFFRACTION99.91
2.21-2.380.18241440.15954334X-RAY DIFFRACTION99.96
2.38-2.620.1741350.16644343X-RAY DIFFRACTION99.69
2.62-30.19871350.17574367X-RAY DIFFRACTION99.93
3-3.780.15821390.15544399X-RAY DIFFRACTION99.71
3.78-31.020.16171420.16274571X-RAY DIFFRACTION99.16
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7053417096730.104762869369-0.2892614286280.8621428600010.1560152651440.422681841012-0.0584290093810.0176612653643-0.1468851912040.0438096672679-0.00730581453642-0.1656288759820.06060268603920.0760188686615-0.01132764509020.1056665738630.035950416176-0.01994560968160.106835810433-0.004679509332340.16744164087915.3479833578-18.1412237795-18.3202435942
20.05040427791180.0141600733720.1122269170140.4145175927970.3072234518660.2522396208070.0149302245997-0.03396131138980.134468600055-0.1113943382810.0308579384008-0.190311464508-0.1652549336510.1848507190591.85225571979E-80.134597124833-0.01777062999580.02135220212570.146827168451-0.008926464904990.15279914471516.87380512329.3425863796-18.7500965623
30.677454281320.169713120049-0.04732674074580.6929198020480.005562155398290.4705535974860.0180944063628-0.09410649505230.09159995935470.140811185779-0.00504099584207-0.00233080313698-0.0480569582863-0.03203224245930.01288265095190.1116686658680.004212818851320.002980429282020.107218970823-0.02937036150350.0850905191675-1.244104283016.72229664881-1.18589187117
40.417736997167-0.4097716327-0.02091262413770.6946608561160.312297210470.796107882289-0.005264072899860.042472387506-0.1848460309360.0456894221131-0.07935690082670.1221567283510.0347285447761-0.0666968042127-0.03708636455250.0785076103506-0.001607304797240.004336092703350.092979591646-0.03345949985480.110459002341-8.40635665154-12.2652855414-12.3055877663
50.03977199054520.207084594741-0.1619061948730.328222986507-0.1572945592730.240891306023-0.00275502040168-0.00422400514935-0.0670589111449-0.1779746605410.02877216999820.02449852011810.00231973464781-0.06722989774720.002786383208650.1401881381340.00674989056676-0.02820710284690.133843341423-0.04465917391440.1017057206722.39157696206-15.2302321575-30.7085176019
60.03294230403120.0008843615937210.03206086342490.07046794211510.06299424641050.06749556101670.04373131404090.0787828176636-0.04849520111720.0240091914173-0.06161686688690.01149768522270.00733122521649-0.00239567966018-5.81521616424E-50.1278289896140.01113318681420.001242405562180.1035667192060.007953460301610.08343660928280.2781286729753.18583899341-17.3165594868
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain A and resid 135:231
2X-RAY DIFFRACTION2chain A and resid 232:289
3X-RAY DIFFRACTION3chain A and resid 290:423
4X-RAY DIFFRACTION4chain A and resid 424:494
5X-RAY DIFFRACTION5chain T or chain D
6X-RAY DIFFRACTION6chain U or chain P

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