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- PDB-8b67: The crystal structure of M644G variant of DNA Pol Epsilon contain... -

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Basic information

Entry
Database: PDB / ID: 8b67
TitleThe crystal structure of M644G variant of DNA Pol Epsilon containing CTP in the polymerase active site
Components
  • DNA polymerase epsilon catalytic subunit A
  • Primer DNA sequence
  • Template DNA sequence
KeywordsDNA BINDING PROTEIN / protein-DNA complex
Function / homology
Function and homology information


gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling ...gene conversion / DNA replication initiation / epsilon DNA polymerase complex / nucleotide-excision repair, DNA gap filling / SUMO binding / DNA replication proofreading / Activation of the pre-replicative complex / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / mitotic sister chromatid cohesion / leading strand elongation / nuclear replication fork / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / base-excision repair / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / mRNA binding / DNA binding / zinc ion binding / nucleus
Similarity search - Function
DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily ...DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / DNA polymerase family B, thumb domain / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
ACETATE ION / CYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA polymerase epsilon catalytic subunit A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
Model detailsThe ternary complex structure of M644G variant of DNA Pol Epsilon with an incoming ribonucleotide ...The ternary complex structure of M644G variant of DNA Pol Epsilon with an incoming ribonucleotide CTP and template DNA
AuthorsParkash, V. / Johansson, E.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Cancerfonden Sweden
Swedish Research Council Sweden
CitationJournal: Nucleic Acids Res. / Year: 2023
Title: A sensor complements the steric gate when DNA polymerase epsilon discriminates ribonucleotides.
Authors: Parkash, V. / Kulkarni, Y. / Bylund, G.O. / Osterman, P. / Kamerlin, S.C.L. / Johansson, E.
History
DepositionSep 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase epsilon catalytic subunit A
P: Primer DNA sequence
T: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,9718
Polymers145,3093
Non-polymers6625
Water27015
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: The wild-type structure of the complex has been solved before (PDB ID 4m8o).
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6850 Å2
ΔGint-59 kcal/mol
Surface area49270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)162.061, 67.360, 152.129
Angle α, β, γ (deg.)90.000, 111.480, 90.000
Int Tables number5
Space group name H-MC121

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA polymerase epsilon catalytic subunit A / 3'-5' exodeoxyribonuclease / DNA polymerase II subunit A


Mass: 137101.453 Da / Num. of mol.: 1 / Fragment: Catalytic subunit of DNA Pol Epsilon / Mutation: M644G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: POL2, DUN2, YNL262W, N0825 / Plasmid: pET 28a / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P21951, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters

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DNA chain , 2 types, 2 molecules PT

#2: DNA chain Primer DNA sequence


Mass: 3293.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Template DNA sequence


Mass: 4914.190 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 20 molecules

#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE / Cytidine triphosphate


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 15 / 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: 2.69 Å3/Da / Density % sol: 54.36 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 10mM Tris, 15% PEG8K,10mM CaCl2

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-3 / Wavelength: 0.9677 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 3, 2018
RadiationMonochromator: Si (111), double crystal monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 2.6→75.4 Å / Num. obs: 47721 / % possible obs: 98.9 % / Redundancy: 3.95 % / Biso Wilson estimate: 77.85 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.135 / Rrim(I) all: 0.157 / Χ2: 1.102 / Net I/σ(I): 6.97
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
2.6-2.94.1011.7930.665458113342133080.3742.06399.7
2.9-3.54.0240.4562.645971614884148400.9330.52699.7
3.5-4.33.7470.1348.9533363913189030.9890.15697.5
4.3-63.8260.06916.6925406679966410.9950.0897.7
6-103.9020.04721.6512275316731460.9980.05599.3
10-40.673.5720.02829.5231549188830.9990.03396.2

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XSCALEdata scaling
PDB_EXTRACT3.27data extraction
MOSFLMdata reduction
PHASER3.55phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4m8o

4m8o


Resolution: 2.6→75.4 Å / SU ML: 0.46 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 30.43 / Stereochemistry target values: ML / Details: Phenix
RfactorNum. reflection% reflection
Rfree0.2755 2332 5 %
Rwork0.2388 44323 -
obs0.2406 46655 98.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 140.65 Å2 / Biso mean: 56.2649 Å2 / Biso min: 14.09 Å2
Refinement stepCycle: final / Resolution: 2.6→75.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8570 527 36 15 9148
Biso mean--37.64 36.73 -
Num. residues----1111
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 17

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.6-2.650.44771360.397725952731100
2.65-2.710.40311570.390625972754100
2.71-2.770.36731350.356526172752100
2.77-2.840.35011410.34112609275099
2.84-2.920.32111270.314426692796100
2.92-3.010.3371390.304725942733100
3.01-3.10.37651440.297725862730100
3.1-3.210.33661260.276926512777100
3.21-3.340.28361320.259826282760100
3.34-3.490.2791330.22882618275199
3.49-3.680.2891400.22762570271098
3.68-3.910.23941500.22872542269297
3.91-4.210.23791300.19642535266595
4.21-4.630.18321240.17992425254991
4.64-5.310.24541430.182826332776100
5.31-6.680.22241380.207526972835100
6.68-75.40.23791370.18742757289499
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)
11.8659-0.59010.37432.17310.50933.20850.00580.61510.0427-0.1439-0.26590.2488-0.0704-0.87890.16560.2371-0.0394-0.01870.65530.02850.3971-27.144513.927811.6121
20.32670.03080.47731.51140.07181.199-0.1335-0.056-0.7894-0.13740.01120.20110.8075-0.0308-0.06340.7149-0.10990.01410.4957-0.05940.5335-11.008-13.84512.6672
31.896-0.08150.07521.29710.75692.83350.0901-0.31510.43990.0965-0.10540.2123-0.3887-0.65880.19880.3068-0.00060.04720.384-0.03520.3985-21.928525.797734.7914
41.5226-0.04420.80250.57980.30842.80310.17870.1283-0.20010.37960.0957-0.08870.52510.9917-0.11860.40220.0314-0.09050.26470.09370.20559.40996.991733.28
51.80580.34840.13041.25610.51312.38-0.01870.33950.0959-0.27640.0375-0.33020.0381.7225-0.12510.0436-0.2313-0.02521.23920.03040.349925.610517.147826.3969
60.81170.29530.11940.62170.07141.48180.1269-0.2435-0.58130.5363-0.18470.00130.59050.1445-0.15320.86510.0856-0.03350.59410.10120.63112.592-13.129443.5374
71.31831.05250.51651.08011.00672.5924-0.00020.0720.3133-0.0973-0.07380.0567-0.32870.5792-0.02930.2528-0.0595-0.04510.46640.02550.34711.498716.035828.9742
81.09960.22770.42360.328-0.11731.8597-0.1760.36880.0885-0.31740.1986-0.2673-0.59871.1203-0.04380.3784-0.13710.03581.12550.06480.429923.224519.5517.3419
92.10790.04530.27171.49880.2362.7816-0.0167-0.43470.20350.30860.01960.1273-0.06720.01180.02670.4599-0.0519-0.05090.3808-0.03220.29173.354222.794561.6111
103.1147-1.7251.53524.6036-4.43546.45360.3764-0.5284-0.6423-0.00650.00830.25760.12060.2748-0.4360.5324-0.0029-0.11750.47080.0470.31516.1167.07353.2648
111.4196-1.3024-0.30162.5572-2.0185.28370.5147-0.0492-0.34440.44030.25620.52720.8061-0.6029-0.5790.4383-0.1053-0.07230.55190.11780.38062.82536.54347.9967
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 29 through 137 )A29 - 137
2X-RAY DIFFRACTION2chain 'A' and (resid 138 through 248 )A138 - 248
3X-RAY DIFFRACTION3chain 'A' and (resid 249 through 511 )A249 - 511
4X-RAY DIFFRACTION4chain 'A' and (resid 512 through 577 )A512 - 577
5X-RAY DIFFRACTION5chain 'A' and (resid 578 through 693 )A578 - 693
6X-RAY DIFFRACTION6chain 'A' and (resid 694 through 768 )A694 - 768
7X-RAY DIFFRACTION7chain 'A' and (resid 769 through 884 )A769 - 884
8X-RAY DIFFRACTION8chain 'A' and (resid 885 through 943 )A885 - 943
9X-RAY DIFFRACTION9chain 'A' and (resid 944 through 1185 )A944 - 1185
10X-RAY DIFFRACTION10chain 'P' and (resid 1 through 10 )P1 - 10
11X-RAY DIFFRACTION11chain 'T' and (resid 2 through 16 )T2 - 16

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