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- PDB-6fwk: The crystal structure of Pol2CORE-M644G in complex with DNA and a... -

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Basic information

Entry
Database: PDB / ID: 6fwk
TitleThe crystal structure of Pol2CORE-M644G in complex with DNA and an incoming nucleotide
Components
  • DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')
  • DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')
  • DNA polymerase epsilon catalytic subunit A
KeywordsDNA BINDING PROTEIN / Pol epsilon / M644G / 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'-DEOXYADENOSINE 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 / molecular replacement / Resolution: 2.503 Å
AuthorsParkash, V. / Johansson, E.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Swedish Research Council Sweden
Cancerfonden Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nat Commun / Year: 2019
Title: Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon.
Authors: Parkash, V. / Kulkarni, Y. / Ter Beek, J. / Shcherbakova, P.V. / Kamerlin, S.C.L. / Johansson, E.
History
DepositionMar 6, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 30, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 6, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _pdbx_seq_map_depositor_info.one_letter_code_mod

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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: DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')
T: DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')
B: DNA polymerase epsilon catalytic subunit A
C: DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')
D: DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)291,28415
Polymers289,9896
Non-polymers1,2949
Water19811
1
A: DNA polymerase epsilon catalytic subunit A
P: DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')
T: DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,6227
Polymers144,9953
Non-polymers6274
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6570 Å2
ΔGint-54 kcal/mol
Surface area49550 Å2
MethodPISA
2
B: DNA polymerase epsilon catalytic subunit A
C: DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')
D: DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,6628
Polymers144,9953
Non-polymers6675
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6570 Å2
ΔGint-71 kcal/mol
Surface area49940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)153.961, 70.342, 158.965
Angle α, β, γ (deg.)90.000, 112.820, 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 / DNA polymerase II subunit A


Mass: 137101.453 Da / Num. of mol.: 2 / Mutation: M644G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: POL2, DUN2, YNL262W, N0825 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P21951, DNA-directed DNA polymerase

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DNA chain , 2 types, 4 molecules PCTD

#2: DNA chain DNA (5'-D(P*TP*AP*AP*CP*CP*GP*CP*GP*TP*TP*(DOC))-3')


Mass: 3293.174 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*TP*CP*TP*TP*GP*AP*AP*CP*GP*CP*GP*GP*TP*TP*A)-3')


Mass: 4599.996 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 20 molecules

#4: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 11 / 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: 2.81 Å3/Da / Density % sol: 56.17 % / Description: plate crystal
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 50mM MES, 150mM NaAc and 8% PEG20K / PH range: 6.5-7.5

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Cryo stream
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 5, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.5→19.988 Å / Num. obs: 108097 / % possible obs: 99.2 % / Redundancy: 3.437 % / Biso Wilson estimate: 64.42 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.091 / Rrim(I) all: 0.108 / Χ2: 1.009 / Net I/σ(I): 8.76 / Num. measured all: 371529 / Scaling rejects: 52
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
2.5-2.573.4431.6580.6325905799475230.3621.96194.1
2.57-2.643.5641.280.8527717778377770.4851.50699.9
2.64-2.713.5351.0271.0726836760975910.6051.2199.8
2.71-2.83.50.8231.3325711736173470.6990.97399.8
2.8-2.893.440.61.8624482713471170.7970.71199.8
2.89-2.993.3380.4922.2722923689668670.8410.58699.6
2.99-3.13.2280.3443.1121499669066600.920.41499.6
3.1-3.233.5730.2534.5722860641763980.9630.29799.7
3.23-3.383.5380.1756.4321856619061780.9830.20799.8
3.38-3.543.5010.1248.9920720593859190.990.14699.7
3.54-3.733.4710.09411.6519317559055650.9920.11299.6
3.73-3.963.2330.07513.7617165534053100.9930.0999.4
3.96-4.233.4040.06117.2416943499449780.9950.07299.7
4.23-4.573.5690.04821.2516689468446760.9970.05799.8
4.57-5.013.4850.04522.0815000432243040.9970.05399.6
5.01-5.63.3480.04421.4513002390238840.9970.05399.5
5.6-6.463.2040.04520.1811097348034640.9970.05499.5
6.46-7.923.490.03823.6710232295229320.9980.04599.3
7.92-11.193.2110.02629.137346230322880.9990.03299.3
11.19-19.9883.2060.02429.124229133913190.9990.02998.5

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.503→19.988 Å / SU ML: 0.4 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 33.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2627 5405 5.02 %
Rwork0.2229 102280 -
obs0.2249 107685 99.2 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 124.48 Å2 / Biso mean: 73.0983 Å2 / Biso min: 36.76 Å2
Refinement stepCycle: final / Resolution: 2.503→19.988 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17293 1058 67 11 18429
Biso mean--53.58 68.34 -
Num. residues----2252
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 (%)
2.503-2.53140.39291660.37252895306186
2.5314-2.56120.41781620.373234773639100
2.5612-2.59230.40021740.360633993573100
2.5923-2.62510.34991660.349134163582100
2.6251-2.65950.36171540.336833933547100
2.6595-2.69590.33451840.330834133597100
2.6959-2.73430.36531870.337533913578100
2.7343-2.7750.40091890.325633843573100
2.775-2.81830.31211840.310934633647100
2.8183-2.86430.32821800.302433783558100
2.8643-2.91360.33741950.298333863581100
2.9136-2.96640.33991860.298934213607100
2.9664-3.02330.37051790.301934123591100
3.0233-3.08480.3691620.30134043566100
3.0848-3.15160.3241710.290834213592100
3.1516-3.22460.30771620.275234593621100
3.2246-3.30490.33921870.267433633550100
3.3049-3.39380.28481970.253434373634100
3.3938-3.49320.25861900.230334023592100
3.4932-3.60530.28111930.222934083601100
3.6053-3.73340.22761580.21934443602100
3.7334-3.88180.28852050.215134043609100
3.8818-4.05710.23121710.19453423359499
4.0571-4.2690.23341680.185934563624100
4.269-4.53350.22181840.174834583642100
4.5335-4.87890.21681900.176134163606100
4.8789-5.36120.22481860.178934843670100
5.3612-6.11750.23862020.19634433645100
6.1175-7.63530.23041890.204534963685100
7.6353-19.98830.19291840.16073534371898

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