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- PDB-4m04: Human DNA Polymerase Mu ternary complex -

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Basic information

Entry
Database: PDB / ID: 4m04
TitleHuman DNA Polymerase Mu ternary complex
Components
  • DNA-directed DNA/RNA polymerase mu
  • downstream primer strand
  • template strandTranscription (biology)
  • upstream primer strand
KeywordsTRANSFERASE/DNA / polymerase / DNA 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 / Beta Polymerase; domain 3 / DNA polymerase, thumb domain / DNA polymerase beta, N-terminal domain-like / 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-directed DNA/RNA polymerase mu / DNA nucleotidylexotransferase (TdT) / DNA-directed DNA/RNA polymerase mu / Beta Polymerase; domain 3 / DNA polymerase, thumb domain / DNA polymerase beta, N-terminal domain-like / 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 / Beta Polymerase, domain 2 / Beta Polymerase; domain 2 / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Nucleotidyltransferase superfamily / 5' to 3' exonuclease, C-terminal subdomain / DNA polymerase; domain 1 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
2'-DEOXYURIDINE 5'-ALPHA,BETA-IMIDO-TRIPHOSPHATE / DNA / DNA-directed DNA/RNA polymerase mu
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.898 Å
AuthorsMoon, A.F. / Pryor, J.M. / Ramsden, D.A. / Kunkel, T.A. / Bebenek, K. / Pedersen, L.C.
CitationJournal: Nat.Struct.Mol.Biol. / Year: 2014
Title: Sustained active site rigidity during synthesis by human DNA polymerase mu.
Authors: Moon, A.F. / Pryor, J.M. / Ramsden, D.A. / Kunkel, T.A. / Bebenek, K. / Pedersen, L.C.
History
DepositionAug 1, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2014Provider: repository / Type: Initial release
Revision 1.1Mar 5, 2014Group: Database references
Revision 1.2Mar 19, 2014Group: Database references
Revision 1.3Nov 15, 2017Group: Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.4Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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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: template strand
P: upstream primer strand
D: downstream primer strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,17713
Polymers45,1784
Non-polymers9999
Water6,864381
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)59.892, 68.467, 109.773
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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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: Polymerase Mu Loop2 deletion variant, UNP residues 132-494
Source method: isolated from a genetically manipulated source
Details: Modified version of the pGEX4T3 vector backbone, where a TEV protease cleavage site has been inserted downstream of the thrombin cleavage site, and the sequence of the multicloning site has ...Details: Modified version of the pGEX4T3 vector backbone, where a TEV protease cleavage site has been inserted downstream of the thrombin cleavage site, and the sequence of the multicloning site has been replaced by that from the pMALX vector (Moon et al, 2010). Three TGA stop codons have been inserted downstream of the MCS, each utilizing a different reading frame.
Source: (gene. exp.) Homo sapiens (human) / Gene: POLM, polmu / Plasmid: pGEXM / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NP87, DNA-directed DNA polymerase

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DNA chain , 3 types, 3 molecules TPD

#2: DNA chain template strand / Transcription (biology)


Mass: 2740.812 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: synthesized by Oligos Etc
#3: DNA chain upstream primer strand


Mass: 1190.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: synthesized by Oligos Etc
#4: DNA chain downstream primer strand


Mass: 1191.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: synthesized by Oligos Etc

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Non-polymers , 7 types, 390 molecules

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


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#8: Chemical ChemComp-CL / CHLORIDE ION / Chloride


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


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#10: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 381 / Source method: isolated from a natural source / Formula: H2O

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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: 2.49 Å3/Da / Density % sol: 50.62 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Crystals were grown by mixing 1uL of protein/DNA mixture with 1uL mother liquor (85-90mM HEPES pH 7.5, 17-18% PEG4000), using the sitting drop vapor diffusion technique. Crystals were then ...Details: Crystals were grown by mixing 1uL of protein/DNA mixture with 1uL mother liquor (85-90mM HEPES pH 7.5, 17-18% PEG4000), using the sitting drop vapor diffusion technique. Crystals were then transferred to a cryoprotectant solution containing 0.1M HEPES pH 7.5, 10mM MgCl2, 50mM NaCl, 20% PEG4000, 5% glycerol, 1mM dUMPNPP in two steps. , VAPOR DIFFUSION, SITTING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 93 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 92 / Detector: CCD / Date: Oct 8, 2012 / Details: VariMaxHF mirrors
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.898→50 Å / Num. all: 36290 / Num. obs: 36290 / % possible obs: 99.7 % / Observed criterion σ(I): -3 / Redundancy: 6.2 % / Biso Wilson estimate: 24.04 Å2 / Rsym value: 0.064 / Χ2: 1.023 / Net I/σ(I): 13.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
1.898-1.973.50.31634900.982198.1
1.97-2.053.90.25635581.096199.4
2.05-2.144.50.19735881.0381100
2.14-2.255.30.17235751.0711100
2.25-2.395.90.14436401.041100
2.39-2.586.60.12336041.0541100
2.58-2.8480.10436451.061100
2.84-3.258.20.06936530.981100
3.25-4.098.10.04836920.9811100
4.09-507.70.04238450.989199.3

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
PHENIX1.8_1069refinement
PDB_EXTRACT3.11data extraction
StructureStudiodata collection
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2IHM (molecule B)

2ihm


Resolution: 1.898→23.174 Å / Occupancy max: 1 / Occupancy min: 0.3 / FOM work R set: 0.8651 / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 19.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.1959 1805 4.99 %random
Rwork0.1639 ---
obs0.1654 36188 99.52 %-
all-36290 --
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 69.43 Å2 / Biso mean: 15.4651 Å2 / Biso min: 1.95 Å2
Refinement stepCycle: LAST / Resolution: 1.898→23.174 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2564 344 56 381 3345
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.013232
X-RAY DIFFRACTIONf_angle_d1.3254483
X-RAY DIFFRACTIONf_chiral_restr0.085491
X-RAY DIFFRACTIONf_plane_restr0.006520
X-RAY DIFFRACTIONf_dihedral_angle_d12.7721036
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8983-1.94960.26471320.20582517264997
1.9496-2.0070.25311340.2042589272399
2.007-2.07170.26571390.184526212760100
2.0717-2.14570.21651370.167726212758100
2.1457-2.23160.21911380.168226372775100
2.2316-2.3330.23461380.168326122750100
2.333-2.45590.21411390.168426412780100
2.4559-2.60960.19081390.178626342773100
2.6096-2.81080.2311370.179326352772100
2.8108-3.0930.21571400.183526592799100
3.093-3.53920.15741400.154526872827100
3.5392-4.45390.13811430.133126932836100
4.4539-23.17620.19721490.159528372986100

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