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- PDB-8b7e: The crystal structure of N828V variant of DNA Pol Epsilon contain... -

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Basic information

Entry
Database: PDB / ID: 8b7e
TitleThe crystal structure of N828V variant of DNA Pol Epsilon containing UTP in the polymerase active site
Components
  • DNA polymerase epsilon catalytic subunit A
  • Primer DNA sequence
  • Template DNA sequence
KeywordsDNA BINDING PROTEIN / protein-DNA complex / ribonucleotide / DNA polymerase active site
Function / homology
Function and homology information


gene conversion / DNA replication initiation / epsilon DNA polymerase complex / SUMO binding / nucleotide-excision repair, DNA gap filling / Activation of the pre-replicative complex / DNA replication proofreading / Termination of translesion DNA synthesis / single-stranded DNA 3'-5' DNA exonuclease activity / mitotic DNA replication checkpoint signaling ...gene conversion / DNA replication initiation / epsilon DNA polymerase complex / SUMO binding / nucleotide-excision repair, DNA gap filling / Activation of the pre-replicative complex / DNA replication proofreading / Termination of translesion DNA synthesis / 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 / Dual incision in TC-NER / error-prone translesion synthesis / base-excision repair, gap-filling / replication fork / base-excision repair / double-strand break repair via nonhomologous end joining / DNA-templated DNA replication / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 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, thumb domain / Zinc finger domain of DNA polymerase-epsilon / 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, exonuclease domain ...: / : / DNA polymerase epsilon catalytic subunit A, thumb domain / Zinc finger domain of DNA polymerase-epsilon / 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, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease 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
URIDINE 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 dTTP 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 29, 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
B: DNA polymerase epsilon catalytic subunit A
C: Primer DNA sequence
D: Template DNA sequence
P: Primer DNA sequence
T: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,72113
Polymers290,5006
Non-polymers1,2217
Water1,47782
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)145,9067
Polymers145,2503
Non-polymers6564
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6800 Å2
ΔGint-44 kcal/mol
Surface area50460 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)145,8146
Polymers145,2503
Non-polymers5643
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6820 Å2
ΔGint-41 kcal/mol
Surface area50110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)154.360, 69.990, 158.320
Angle α, β, γ (deg.)90.000, 112.550, 90.000
Int Tables number3
Space group name H-MP121

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Components

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Protein , 1 types, 2 molecules AB

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


Mass: 137058.594 Da / Num. of mol.: 2 / Fragment: Catalytic subunit of DNA Pol Epsilon / Mutation: N828V
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, 4 molecules CPDT

#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: 4898.191 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 89 molecules

#4: Chemical ChemComp-UTP / URIDINE 5'-TRIPHOSPHATE


Mass: 484.141 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H15N2O15P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: UTP*YM
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


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


Mass: 18.015 Da / Num. of mol.: 82 / 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: 2.72 Å3/Da / Density % sol: 54.85 %
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 EIGER X 4M / 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→78.39 Å / Num. obs: 95522 / % possible obs: 98.9 % / Redundancy: 4.3 % / Biso Wilson estimate: 48.28 Å2 / CC1/2: 0.994 / Rmerge(I) obs: 0.166 / Rpim(I) all: 0.089 / Rrim(I) all: 0.189 / Net I/σ(I): 6.8 / Num. measured all: 405993 / Scaling rejects: 271
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.6-2.644.41.2452071746810.3230.6431.4061.499.9
14.24-78.393.50.04422386420.9860.0290.05313.398.2

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Processing

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

4m8o


Resolution: 2.6→26.75 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 25.32 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2301 4765 5 %
Rwork0.1978 90559 -
obs0.1995 95324 98.66 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 126.64 Å2 / Biso mean: 57.5937 Å2 / Biso min: 20.65 Å2
Refinement stepCycle: final / Resolution: 2.6→26.75 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17622 1052 68 82 18824
Biso mean--41.23 43.6 -
Num. residues----2292
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 (%)
2.6-2.630.36291660.332329763142100
2.63-2.660.34951690.314730363205100
2.66-2.690.35081590.30530593218100
2.69-2.730.33031510.304829853136100
2.73-2.760.3161500.289330363186100
2.76-2.80.33991500.292430833233100
2.8-2.840.31251490.279830053154100
2.84-2.880.31571590.2730473206100
2.88-2.930.33061560.274930833239100
2.93-2.980.31931390.272430173156100
2.98-3.030.29671650.264630533218100
3.03-3.080.2921530.253729983151100
3.08-3.140.29211480.23830623210100
3.14-3.210.27471620.227730523214100
3.21-3.270.23741690.227629823151100
3.27-3.350.24971690.221530483217100
3.35-3.430.24391570.211730213178100
3.43-3.530.2571480.19793089323799
3.53-3.630.22741680.18482939310798
3.63-3.750.22411480.182973312197
3.75-3.880.19791480.17692950309896
3.88-4.040.19711570.16482891304895
4.04-4.220.17141380.15832792293091
4.22-4.440.14911340.14282859299392
4.44-4.720.1791650.14043054321999
4.72-5.080.19171780.152530623240100
5.08-5.590.1891720.162230773249100
5.59-6.380.2041920.177230813273100
6.39-8.010.23031670.182931193286100
8.01-26.750.19451790.16173130330998
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)
11.6243-0.70140.22681.807-0.16911.87010.14180.5022-0.0037-0.5431-0.05820.07940.0439-0.0126-0.06940.49790.02890.02590.41430.03990.280522.1645-13.4017-19.7452
20.1706-0.137-0.11481.13870.29360.52430.02190.17830.2921-0.1053-0.07670.0686-0.2616-0.16490.05670.41370.0251-0.02440.29170.09770.42716.07469.2382-5.8489
30.6231-0.02080.31930.3254-0.16140.7034-0.0140.1239-0.039-0.0891-0.0515-0.19870.16630.21980.05420.34210.03790.08960.27020.05450.369640.2818-21.19441.3516
40.48770.0105-0.00751.7461-0.67571.2372-0.0907-0.22030.05010.0524-0.0124-0.2708-0.1382-0.11650.0440.36320.0684-0.01040.3577-0.05360.347624.8264-4.878428.2468
50.9605-0.17160.29130.5822-0.39130.99620.0128-0.0961-0.0872-0.0089-0.022-0.10730.043-0.04930.00870.3208-0.01920.02160.24430.02830.316623.0734-15.068721.0399
61.5329-0.19640.24210.52450.07660.67560.0108-0.07770.00590.11930.0437-0.23440.07970.0627-0.03550.3310.0126-0.05630.27470.02380.3644.6836-20.205228.8535
71.18930.75370.63011.36170.56180.96320.1253-0.59190.15140.059-0.3294-0.0981-0.2827-0.22440.06960.3427-0.0416-0.09240.42190.01220.438445.7615-5.633827.4863
81.81570.13790.55140.61060.98510.8267-0.1546-0.00510.1391-0.08820.1112-0.4535-0.28750.1572-0.00240.4065-0.0768-0.05740.29780.02040.539542.4898-4.889722.5524
91.19740.2071-0.02511.38620.01581.59690.1556-0.55360.11640.5473-0.0908-0.1778-0.00060.2343-0.08980.4768-0.004-0.00390.5138-0.0320.284101.848821.466594.6455
102.0782-0.16280.23131.515-0.2641.401-0.0051-0.20440.48720.1602-0.0422-0.3098-0.30840.31940.03280.3866-0.0334-0.00910.4441-0.09770.4421109.746338.2182.1523
110.07210.3181-0.29631.17740.20790.9110.045-0.0620.2141-0.0128-0.07920.041-0.35490.10470.04180.40820.036-0.01530.3249-0.05080.4297104.425441.346979.8465
121.73280.1230.22140.9398-0.19561.61720.0059-0.1182-0.2010.08690.05250.11590.2845-0.3946-0.03580.4428-0.08290.07310.41760.01290.332378.64798.67684.2584
130.6219-0.14810.2698-0.0780.04410.9459-0.06770.03690.0379-0.0226-0.03560.1130.1489-0.13920.07140.3474-0.00230.01870.2865-0.05220.355188.321316.41363.3516
140.87950.37630.76350.62180.1521.58660.02230.7802-0.1432-0.09910.1723-0.2010.11820.8699-0.00790.41990.12680.03641.0615-0.16430.4223110.19811.686738.1504
151.1295-0.00740.12621.02220.64221.4107-0.02530.38020.1257-0.1183-0.20010.403-0.2537-0.03690.14180.3369-0.0121-0.08060.41220.02440.498991.662635.75955.0531
161.2082-0.03770.6790.58610.48831.19150.07980.5187-0.1436-0.08170.007-0.04530.05820.4735-0.03210.30190.08690.02960.5452-0.04850.2995105.579515.855248.2945
171.9339-0.4512-0.09730.60490.11171.2322-0.02350.0684-0.0656-0.08860.02990.25150.0767-0.1263-0.01040.34610.0058-0.06970.3882-0.05410.395867.747614.103346.3481
180.9613-0.96520.78481.0974-0.85311.03220.12230.30030.2065-0.2674-0.31510.0094-0.19380.29640.11010.32070.0712-0.08380.4758-0.02190.478977.253128.755947.123
191.7814-0.23740.50950.8287-0.63691.0499-0.17690.07220.05640.00630.07860.2885-0.47-0.23170.12630.36540.1179-0.10260.4042-0.0440.521680.773529.423452.0326
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 31 through 137 )A31 - 137
2X-RAY DIFFRACTION2chain 'A' and (resid 138 through 274 )A138 - 274
3X-RAY DIFFRACTION3chain 'A' and (resid 275 through 577 )A275 - 577
4X-RAY DIFFRACTION4chain 'A' and (resid 578 through 746 )A578 - 746
5X-RAY DIFFRACTION5chain 'A' and (resid 747 through 875 )A747 - 875
6X-RAY DIFFRACTION6chain 'A' and (resid 876 through 1186 )A876 - 1186
7X-RAY DIFFRACTION7chain 'P' and (resid 1 through 10 )P1 - 10
8X-RAY DIFFRACTION8chain 'T' and (resid 2 through 16 )T2 - 16
9X-RAY DIFFRACTION9chain 'B' and (resid 28 through 113 )B28 - 113
10X-RAY DIFFRACTION10chain 'B' and (resid 114 through 195 )B114 - 195
11X-RAY DIFFRACTION11chain 'B' and (resid 196 through 272 )B196 - 272
12X-RAY DIFFRACTION12chain 'B' and (resid 273 through 417 )B273 - 417
13X-RAY DIFFRACTION13chain 'B' and (resid 418 through 577 )B418 - 577
14X-RAY DIFFRACTION14chain 'B' and (resid 578 through 677 )B578 - 677
15X-RAY DIFFRACTION15chain 'B' and (resid 678 through 802 )B678 - 802
16X-RAY DIFFRACTION16chain 'B' and (resid 803 through 943 )B803 - 943
17X-RAY DIFFRACTION17chain 'B' and (resid 944 through 1186 )B944 - 1186
18X-RAY DIFFRACTION18chain 'C' and (resid 1 through 10 )C1 - 10
19X-RAY DIFFRACTION19chain 'D' and (resid 2 through 16 )D2 - 16

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