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- PDB-8b77: The crystal structure of N828V variant of DNA Pol Epsilon contain... -

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Basic information

Entry
Database: PDB / ID: 8b77
TitleThe crystal structure of N828V variant of DNA Pol Epsilon containing dATP in the polymerase active site
Components
  • DNA polymerase epsilon catalytic subunit A
  • Primer DNA sequence
  • Template DNA sequence
KeywordsDNA BINDING PROTEIN / protein-DNA complex / N828V / ribonucleotide
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 / Resolution: 2.7 Å
Model detailsThe ternary complex structure of N828V variant of DNA Pol Epsilon with an incoming dATP 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
P: Primer DNA sequence
T: Template DNA sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,7725
Polymers145,2413
Non-polymers5312
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: The wild type structure of the ternary complex has been solved before (PDB ID 4m8o).
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6370 Å2
ΔGint-36 kcal/mol
Surface area51820 Å2
MethodPISA
Unit cell
Length a, b, c (Å)158.994, 71.030, 155.255
Angle α, β, γ (deg.)90.000, 112.990, 90.000
Int Tables number5
Space group name H-MC121

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Components

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


Mass: 137058.594 Da / Num. of mol.: 1 / 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
#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: 4889.177 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / Deoxyadenosine triphosphate


Mass: 491.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
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: 2.79 Å3/Da / Density % sol: 55.92 %
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: 2.7→78.8 Å / Num. obs: 43465 / % possible obs: 98.6 % / Redundancy: 4.2 % / CC1/2: 0.993 / Rrim(I) all: 0.14 / Net I/σ(I): 4.5
Reflection shellResolution: 2.7→2.8 Å / Num. unique obs: 4612 / CC1/2: 0.42 / Rrim(I) all: 0.79 / % possible all: 99.7

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Processing

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

4m8o


Resolution: 2.7→65.49 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 32.43 / Stereochemistry target values: ML / Details: Phenix
RfactorNum. reflection% reflection
Rfree0.2731 2175 5.01 %
Rwork0.2363 41241 -
obs0.2381 43416 98.32 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 177.3 Å2 / Biso mean: 82.7567 Å2 / Biso min: 28.25 Å2
Refinement stepCycle: final / Resolution: 2.7→65.49 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8764 529 31 0 9324
Biso mean--45.24 --
Num. residues----1136
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 16

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7-2.760.40231450.39625812726100
2.76-2.820.38111480.379225752723100
2.82-2.890.40931370.363126212758100
2.89-2.970.32721450.340725642709100
2.97-3.060.3711340.324625682702100
3.06-3.160.3761400.30052608274899
3.16-3.270.35751330.28652595272899
3.27-3.40.31211490.27322596274599
3.4-3.560.29181160.24352593270999
3.56-3.740.32181140.22932630274499
3.74-3.980.26391300.2172610274099
3.98-4.290.21591490.20182541269098
4.29-4.720.20141350.17542360249590
4.72-5.40.22391300.18242425255592
5.4-6.80.2411380.212726482786100
6.8-65.490.22031320.19252726285899
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)
12.4524-0.4621-0.12313.95751.11772.4674-0.05330.207-0.3689-0.476-0.13250.64020.5173-0.66540.15140.6357-0.1796-0.0420.5503-0.04110.4956-17.6358-0.589312.8999
21.80240.20640.23981.12820.51653.9416-0.0992-0.32760.35670.0388-0.05350.2461-0.2916-0.07670.12570.4477-0.02-0.00610.2438-0.01160.576-6.532121.661936.0528
32.41040.67340.5032.366-0.07430.63430.15290.2062-0.68030.8427-0.2225-0.37241.04530.17030.04891.36870.3658-0.03410.8791-0.09680.705210.5666-12.240536.4794
41.39430.45440.62141.65450.8954.3142-0.0229-0.31820.1870.31210.0447-0.0654-0.05340.7765-0.02180.38070.077-0.02480.5832-0.01940.49848.139119.482547.3346
51.806-0.2712-0.512.672-1.07792.03360.1315-0.4527-0.31860.6101-0.286-0.30450.87461.24080.16521.11820.1643-0.00940.93990.07110.47387.49664.821553.4919
61.2954-1.0731-0.00261.9302-0.62.99860.4435-0.819-0.44570.8302-0.14780.35371.16960.2644-0.32371.09920.0489-0.0140.85160.11030.49713.81964.323148.0273
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 31 through 274 )A31 - 274
2X-RAY DIFFRACTION2chain 'A' and (resid 275 through 658 )A275 - 658
3X-RAY DIFFRACTION3chain 'A' and (resid 659 through 768 )A659 - 768
4X-RAY DIFFRACTION4chain 'A' and (resid 769 through 1186 )A769 - 1186
5X-RAY DIFFRACTION5chain 'P' and (resid 1 through 10 )P1 - 10
6X-RAY DIFFRACTION6chain 'T' and (resid 2 through 16 )T2 - 16

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