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- PDB-8b6k: The crystal structure of M644G variant of DNA Pol Epsilon contain... -

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Basic information

Entry
Database: PDB / ID: 8b6k
TitleThe crystal structure of M644G variant of DNA Pol Epsilon containing dCTP 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
2'-DEOXYCYTIDINE-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: 2.5 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 27, 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
B: DNA polymerase epsilon catalytic subunit A
C: Primer DNA sequence
D: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,42810
Polymers290,4146
Non-polymers1,0144
Water0
1
A: DNA polymerase epsilon catalytic subunit A
P: Primer DNA sequence
T: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,7145
Polymers145,2073
Non-polymers5072
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6380 Å2
ΔGint-32 kcal/mol
Surface area50210 Å2
MethodPISA
2
B: DNA polymerase epsilon catalytic subunit A
C: Primer DNA sequence
D: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,7145
Polymers145,2073
Non-polymers5072
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6390 Å2
ΔGint-29 kcal/mol
Surface area50160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)154.370, 70.420, 158.659
Angle α, β, γ (deg.)90.000, 112.690, 90.000
Int Tables number3
Space group name H-MP121

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Components

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


Mass: 136999.406 Da / Num. of mol.: 2 / Fragment: Catalytic subunit of DNA Pol Epsilon / Mutation: M644G, D290A, E292A
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
#2: DNA chain Primer DNA sequence


Mass: 3293.174 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Template DNA sequence


Mass: 4914.190 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / Deoxycytidine triphosphate


Mass: 467.157 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H16N3O13P3 / Feature type: SUBJECT OF INVESTIGATION
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: 2.76 Å3/Da / Density % sol: 55.45 %
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: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9125 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: 0.9125 Å / Relative weight: 1
ReflectionResolution: 2.5→86.72 Å / Num. obs: 109188 / % possible obs: 99.9 % / Redundancy: 3.9 % / Biso Wilson estimate: 42.4 Å2 / CC1/2: 0.984 / Rmerge(I) obs: 0.2 / Net I/σ(I): 5.4
Reflection shellResolution: 2.5→2.54 Å / Redundancy: 3.6 % / Rmerge(I) obs: 1.26 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 5329 / CC1/2: 0.08 / % possible all: 99.1

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
PDB_EXTRACT3.27data extraction
XDSv2021data reduction
XSCALEv2021data scaling
PHASER3.55phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4m8o

4m8o


Resolution: 2.5→86.72 Å / SU ML: 0.45 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 30.69 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2598 5660 5.19 %
Rwork0.2281 103351 -
obs0.2297 109011 99.55 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 140.81 Å2 / Biso mean: 59.3961 Å2 / Biso min: 19.41 Å2
Refinement stepCycle: final / Resolution: 2.5→86.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17580 1062 58 0 18700
Biso mean--34.57 --
Num. residues----2266
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.5-2.530.41211670.39633428359598
2.53-2.560.41871830.382633773560100
2.56-2.590.37531970.373134443641100
2.59-2.620.36951630.348534443607100
2.62-2.660.35951730.352734223595100
2.66-2.690.36261690.34634773646100
2.69-2.730.39952080.340433823590100
2.73-2.770.35882100.330733953605100
2.77-2.820.34111900.31363432362299
2.82-2.860.34621790.316734243603100
2.86-2.910.36851960.307834293625100
2.91-2.960.35311650.310234433608100
2.96-3.020.3471950.294234453640100
3.02-3.080.34092180.293334143632100
3.08-3.150.32771770.281234453622100
3.15-3.220.30411880.27263454364299
3.22-3.30.32171930.266333833576100
3.3-3.390.3171850.25693450363599
3.39-3.490.28571530.2313464361799
3.49-3.610.2831890.217834653654100
3.61-3.730.24841960.211234243620100
3.73-3.880.24642090.198934423651100
3.88-4.060.21771970.182934583655100
4.06-4.270.18981730.171534673640100
4.27-4.540.19122090.151534533662100
4.54-4.890.16671990.14943433363299
4.89-5.390.16021780.159735273705100
5.39-6.160.22341950.179835013696100
6.16-7.770.20462010.1935393740100
7.77-86.720.16722050.16063490369596
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)
11.33360.33270.54231.08480.41861.6628-0.0198-0.02960.078-0.0024-0.10930.40720.0588-0.19070.09870.25410.01730.01670.1773-0.0360.403313.3475-9.539760.6663
23.5732-2.9924-0.40255.0284-1.70531.70920.04380.2490.2847-0.36820.02130.3679-0.2310.061-0.0680.385-0.0113-0.17090.4567-0.1170.61160.636-0.985645.0817
34.7444-1.8108-2.65347.30923.5419.37640.09120.8565-0.7103-0.6049-0.38870.22240.6978-0.49880.28870.2652-0.0223-0.01290.3572-0.29620.655415.1562-11.351847.8075
46.4731-1.69822.24942.9601-2.89963.1068-0.05260.05530.32310.1255-0.10490.9741-0.4842-0.67860.09790.37820.1196-0.04780.3528-0.09210.68034.2147-0.406550.6384
51.8488-0.37520.7920.8803-0.22951.9681-0.0184-0.15710.01940.102-0.0142-0.11860.0985-0.02650.02820.2409-0.0565-0.00610.23780.01210.2857-43.514624.678511.5184
65.36823.89811.84984.18542.05553.6292-0.2495-0.25710.30310.12670.0314-0.2022-0.489-0.01860.19810.3314-0.0175-0.12570.47130.0750.5917-31.586932.781226.4719
76.72291.61341.90813.56692.15583.8717-0.3932-0.01910.6427-0.0405-0.023-0.6276-0.76380.67390.37220.31-0.1026-0.06350.38720.03060.4487-34.155634.19721.1811
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'A' and resid 30 through 1185)A30 - 1185
2X-RAY DIFFRACTION2(chain 'P' and resid 1 through 10)P1 - 10
3X-RAY DIFFRACTION3(chain 'P' and resid 11 through 11)P11
4X-RAY DIFFRACTION4(chain 'T' and resid 2 through 16)T2 - 16
5X-RAY DIFFRACTION5(chain 'B' and resid 31 through 1185)B31 - 1185
6X-RAY DIFFRACTION6(chain 'C' and resid 1 through 11)C1 - 11
7X-RAY DIFFRACTION7(chain 'D' and resid 2 through 16)D2 - 16

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