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- PDB-6wie: Post-catalytic nicked complex of human Polymerase Mu on a complem... -

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Basic information

Entry
Database: PDB / ID: 6wie
TitlePost-catalytic nicked 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


somatic hypermutation of immunoglobulin genes / B cell differentiation / 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 family X, binding site / DNA polymerase family X signature. / DNA polymerase lambda lyase domain superfamily / DNA polymerase family X / DNA polymerase beta, thumb domain / DNA polymerase, thumb domain superfamily / DNA polymerase beta thumb / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Nucleotidyltransferase superfamily
Similarity search - Domain/homology
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)46,38416
Polymers45,4375
Non-polymers94711
Water6,251347
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6770 Å2
ΔGint-86 kcal/mol
Surface area15810 Å2
Unit cell
Length a, b, c (Å)60.073, 62.247, 118.318
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, 358 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#8: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#9: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#10: Chemical ChemComp-PPV / PYROPHOSPHATE


Mass: 177.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H4O7P2
#11: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#12: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


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


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#14: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 347 / 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: 49.47 %
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: 71986 / % possible obs: 99.8 % / Redundancy: 6.3 % / Biso Wilson estimate: 15.64 Å2 / CC1/2: 0.995 / CC star: 0.999 / Rpim(I) all: 0.047 / Rrim(I) all: 0.116 / Rsym value: 0.106 / Χ2: 0.907 / Net I/σ(I): 18.5
Reflection shellResolution: 1.5→1.53 Å / Redundancy: 5.4 % / Mean I/σ(I) obs: 1.77 / Num. unique obs: 3544 / CC1/2: 0.807 / CC star: 0.945 / Rpim(I) all: 0.266 / Rrim(I) all: 0.636 / Rsym value: 0.575 / Χ2: 0.307 / % possible all: 99.8

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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→34.9 Å / SU ML: 0.1384 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 17.0263
RfactorNum. reflection% reflectionSelection details
Rfree0.1868 2217 3.08 %random
Rwork0.1699 ---
obs0.1704 71906 99.7 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 21.57 Å2
Refinement stepCycle: LAST / Resolution: 1.5→34.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2564 361 52 347 3324
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00863265
X-RAY DIFFRACTIONf_angle_d1.03024519
X-RAY DIFFRACTIONf_chiral_restr0.0731489
X-RAY DIFFRACTIONf_plane_restr0.0069527
X-RAY DIFFRACTIONf_dihedral_angle_d12.97921029
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.530.25971230.22494205X-RAY DIFFRACTION97.92
1.53-1.570.21511530.19814305X-RAY DIFFRACTION99.93
1.57-1.60.21951380.18354315X-RAY DIFFRACTION99.91
1.6-1.650.19541350.17394320X-RAY DIFFRACTION99.93
1.65-1.70.1991330.16844314X-RAY DIFFRACTION99.87
1.7-1.750.18261440.16384308X-RAY DIFFRACTION99.89
1.75-1.810.20011430.16514325X-RAY DIFFRACTION99.84
1.81-1.890.20321320.15934354X-RAY DIFFRACTION99.96
1.89-1.970.18361370.16724323X-RAY DIFFRACTION99.96
1.97-2.080.18271420.16844363X-RAY DIFFRACTION100
2.08-2.210.1841390.16534344X-RAY DIFFRACTION100
2.21-2.380.19281380.16494358X-RAY DIFFRACTION99.96
2.38-2.620.1951430.17324395X-RAY DIFFRACTION99.63
2.62-2.990.20081340.17924417X-RAY DIFFRACTION99.85
2.99-3.770.17191400.16314442X-RAY DIFFRACTION99.63
3.77-34.90.16871430.16814601X-RAY DIFFRACTION99.06
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)
10.7124425533210.0133370058166-0.1643772082980.7703875111630.03819574735170.389029591247-0.03745978063250.0174190472143-0.1467567351740.0386622532763-0.013802278002-0.1762842228080.04896585298180.0809973748602-0.02233437563510.1119701385630.0317996349047-0.02125380325980.115123292836-0.01349822824920.18401979470215.0431672083-18.1967767838-18.1210592298
20.0928669498061-0.03336732726940.1993892187450.4331017010970.2511515200290.236260022487-0.0035121292332-0.05260657506170.108746206331-0.1327410314230.0369183269047-0.176425301454-0.2387551649640.2322452744191.02887239961E-80.165502168637-0.02990822582310.0208180787430.168098334448-0.009898026217420.16384642966416.89102357339.59164644749-19.1266291064
30.6649155206840.192279378059-0.0232402855890.656445419759-0.02842652647930.3990289692610.0226582752749-0.09318659190180.09260635042390.162385075836-0.00691605391229-0.00269176538318-0.058952168383-0.008305407486470.0003735980359380.129827753671-0.001727988016880.001420311715980.122866417344-0.03065952207430.102310480481-0.6405049914686.76301428969-1.18014520246
40.371492066216-0.236795151535-0.0006398051981680.7575931153320.2547409981160.849601142446-8.91932485041E-50.0227132222891-0.1670671348350.0442258772916-0.06100046214540.1032129410390.0356520472357-0.0534096009172-0.06432808254310.0956724211907-0.001348136140510.002483239858760.11403560738-0.02936196869420.124655804247-8.06242639622-12.4207944653-12.0959524744
50.0341437399550.136631312148-0.1171379466220.229709101164-0.1255660047540.1256857794470.0297590497543-0.045929920489-0.0903467945997-0.227329123261-0.009931538240820.0403242974276-0.0603638231356-0.0459113533036-0.001356452187680.1605584738630.0143595759692-0.02837877363430.140939253717-0.04224336547330.1173017799012.39606560682-15.3098642848-30.7163694641
60.04966305899480.02911717760820.05438271125230.07027189538330.07058194391060.06203736526870.1543893751870.119229292211-0.05926909328880.0445435390362-0.1205229973150.0548839555427-0.03269996021240.03852953989122.00754782155E-60.1495608133280.00884177979337-0.004825015847620.1227501002010.003330105032930.09537301072850.1088888467273.52808791133-17.7408113348
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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