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- PDB-8b76: The crystal structure of M644G variant of DNA Pol Epsilon contain... -

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Basic information

Entry
Database: PDB / ID: 8b76
TitleThe crystal structure of M644G variant of DNA Pol Epsilon containing dTTP 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 / THYMIDINE-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 UTP 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 28, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release

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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)291,90916
Polymers290,5866
Non-polymers1,32310
Water95553
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)145,9548
Polymers145,2933
Non-polymers6615
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6890 Å2
ΔGint-54 kcal/mol
Surface area50330 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)145,9548
Polymers145,2933
Non-polymers6615
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6820 Å2
ΔGint-52 kcal/mol
Surface area50200 Å2
MethodPISA
Unit cell
Length a, b, c (Å)154.160, 69.880, 159.050
Angle α, β, γ (deg.)90.000, 112.630, 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: 137101.453 Da / Num. of mol.: 2 / 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, 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, 63 molecules

#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate


Mass: 482.168 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 53 / 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: ID29 / Wavelength: 0.97625 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: 0.97625 Å / Relative weight: 1
ReflectionResolution: 2.6→86.83 Å / Num. obs: 96267 / % possible obs: 99.5 % / Redundancy: 3 % / Biso Wilson estimate: 54.57 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.092 / Rpim(I) all: 0.062 / Rrim(I) all: 0.111 / Net I/σ(I): 7.4 / Num. measured all: 291431 / Scaling rejects: 298
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
2.6-2.643.10.6841477147430.4160.4560.8251.599.8
14.24-86.8330.03718236040.9820.0270.04616.891.7

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Processing

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

4m8o


Resolution: 2.6→86.83 Å / SU ML: 0.4 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 28.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2468 4801 5 %
Rwork0.2203 91314 -
obs0.2217 96115 99.24 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 122.31 Å2 / Biso mean: 63.7417 Å2 / Biso min: 22.43 Å2
Refinement stepCycle: final / Resolution: 2.6→86.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17709 1052 72 53 18886
Biso mean--48.01 47.09 -
Num. residues----2291
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 (%)
2.6-2.630.37841700.359330053175100
2.63-2.660.39431720.35313017318999
2.66-2.690.38521470.3443023317099
2.69-2.730.33071590.33413009316899
2.73-2.760.35511490.31333024317399
2.76-2.80.32111520.30633070322299
2.8-2.840.34961440.29622971311599
2.84-2.880.32891480.2843018316699
2.88-2.930.33611630.282430523215100
2.93-2.980.33961400.291630163156100
2.98-3.030.33841650.280330703235100
3.03-3.080.29441580.28032978313699
3.08-3.140.30211470.27153059320699
3.14-3.210.30041550.25933073322899
3.21-3.280.28461730.26142973314699
3.28-3.350.30171700.24553061323199
3.35-3.440.24811560.22582991314799
3.44-3.530.23991450.22593056320198
3.53-3.630.25331680.217330243192100
3.63-3.750.25821520.216630623214100
3.75-3.880.22261520.221731073259100
3.88-4.040.22841670.191230253192100
4.04-4.220.19741480.178130713219100
4.22-4.450.16711560.171130753231100
4.45-4.720.20741650.16763052321799
4.72-5.090.20571770.178230703247100
5.09-5.60.20121690.180630763245100
5.6-6.410.22591970.201330833280100
6.41-8.080.22721620.199931083270100
8.08-86.830.2081750.18093095327095

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