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- PDB-2oq4: Crystal structure of the DNA repair enzyme endonuclease-VIII (Nei... -

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Basic information

Entry
Database: PDB / ID: 2oq4
TitleCrystal structure of the DNA repair enzyme endonuclease-VIII (Nei) from E. coli (E2Q) in complex with AP-site containing DNA substrate
Components
  • 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'
  • 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'
  • Endonuclease VIII
KeywordsHYDROLASE/DNA / Endonuclease VIII / oxidative damage / DNA repair / base excision / covalent intermediate / reaction mechanism / HYDROLASE-DNA COMPLEX
Function / homology
Function and homology information


oxidized pyrimidine nucleobase lesion DNA N-glycosylase activity / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / DNA-(apurinic or apyrimidinic site) endonuclease activity / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / base-excision repair / damaged DNA binding / zinc ion binding
Similarity search - Function
Endonuclease VIII / Nei, N-terminal / Zinc finger, DNA glycosylase/AP lyase-type / Zinc finger, FPG/IleRS-type / DNA glycosylase/AP lyase, zinc finger domain, DNA-binding site / Zinc finger found in FPG and IleRS / Zinc finger FPG-type signature. / Zinc finger FPG-type profile. / MutM-like, N-terminal / Formamidopyrimidine-DNA glycosylase H2TH domain ...Endonuclease VIII / Nei, N-terminal / Zinc finger, DNA glycosylase/AP lyase-type / Zinc finger, FPG/IleRS-type / DNA glycosylase/AP lyase, zinc finger domain, DNA-binding site / Zinc finger found in FPG and IleRS / Zinc finger FPG-type signature. / Zinc finger FPG-type profile. / MutM-like, N-terminal / Formamidopyrimidine-DNA glycosylase H2TH domain / N-terminal domain of MutM-like DNA repair proteins / Formamidopyrimidine-DNA glycosylase N-terminal domain / Formamidopyrimidine-DNA glycosylase N-terminal domain / MutM-like, N-terminal / Formamidopyrimidine-DNA glycosylase, catalytic domain / Formamidopyrimidine-DNA glycosylase catalytic domain profile. / Formamidopyrimidine-DNA glycosylase H2TH domain / DNA glycosylase/AP lyase, H2TH DNA-binding / Helicase, Ruva Protein; domain 3 - #50 / Helicase, Ruva Protein; domain 3 / Ribosomal protein S13-like, H2TH / Alpha-Beta Barrel / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / Endonuclease 8
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsGolan, G. / Zharkov, D.O. / Grollman, A.P. / Shoahm, G.
CitationJournal: To be Published
Title: Active site plasticity of endonuclease VIII: Conformational changes compensating for different substrates and mutations of critical residues
Authors: Golan, G. / Zharkov, D.O. / Grollman, A.P. / Shoham, G.
History
DepositionJan 31, 2007Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 26, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 10, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'
D: 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'
E: 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'
F: 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'
A: Endonuclease VIII
B: Endonuclease VIII
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,73713
Polymers75,2726
Non-polymers4657
Water1,67593
1
C: 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'
D: 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'
A: Endonuclease VIII
hetero molecules


Theoretical massNumber of molelcules
Total (without water)37,7985
Polymers37,6363
Non-polymers1612
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'
F: 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'
B: Endonuclease VIII
hetero molecules


Theoretical massNumber of molelcules
Total (without water)37,9408
Polymers37,6363
Non-polymers3045
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)107.130, 107.130, 164.860
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

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DNA chain , 2 types, 4 molecules CEDF

#1: DNA chain 5'-D(*G*GP*CP*TP*TP*CP*AP*TP*CP*CP*TP*GP*G)-3'


Mass: 3958.571 Da / Num. of mol.: 2 / Source method: obtained synthetically
#2: DNA chain 5'-D(*C*CP*AP*GP*GP*AP*(PED)P*GP*AP*AP*GP*CP*C)-3'


Mass: 3863.531 Da / Num. of mol.: 2 / Source method: obtained synthetically

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Protein , 1 types, 2 molecules AB

#3: Protein Endonuclease VIII / DNA glycosylase/AP lyase Nei / DNA-apurinic or apyrimidinic site / lyase Nei


Mass: 29814.010 Da / Num. of mol.: 2 / Mutation: E2Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: Nei / Plasmid: PET13A / Production host: Escherichia coli (E. coli) / Strain (production host): B834(DE3)
References: UniProt: P50465, Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds, DNA-(apurinic or apyrimidinic site) lyase

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Non-polymers , 4 types, 100 molecules

#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 93 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsSEQUENCING AND CRYSTALLOGRAPHY CONFIRM THE SEQUENCE AS THR A 34, THR B 34, ARG A 112, ARG B 112.

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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: 3.11 Å3/Da / Density % sol: 60.49 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6
Details: 1.8M ammonium sulfate, 0.1M sodium acetate pH 4.6-5.0, VAPOR DIFFUSION, HANGING DROP, temperature 293K
Components of the solutions
IDNameCrystal-IDSol-ID
1ammonium sulfate11
2sodium acetate11
3h2o11
4ammonium sulfate12
5sodium acetate12
6h2o12

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 5ID-B / Wavelength: 1.102 Å
DetectorType: MAR CCD 165 mm / Detector: CCD
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.102 Å / Relative weight: 1
ReflectionResolution: 2.6→16.73 Å / Num. obs: 30010 / % possible obs: 99.6 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 6.4 % / Biso Wilson estimate: 48.1 Å2 / Rmerge(I) obs: 0.083 / Net I/σ(I): 13.1
Reflection shellResolution: 2.6→2.69 Å / Rmerge(I) obs: 0.338 / % possible all: 98.4

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Processing

Software
NameVersionClassification
CNS1refinement
d*TREKdata reduction
d*TREKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2EA0

2ea0


Resolution: 2.6→16 Å / Rfactor Rfree error: 0.008 / Data cutoff high absF: 1272101.92 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
Details: Data processing indicated that the crystal was twinned but the exact nature of the twining was not clear. Treatment of the data with the twinning utility of CNS indicated unequal (major and ...Details: Data processing indicated that the crystal was twinned but the exact nature of the twining was not clear. Treatment of the data with the twinning utility of CNS indicated unequal (major and minor) twinning fractions, but a refinement of the initial model, ignoring the twinning and considering only the major twinning fraction, gave better structural results, with relatively reasonable final R and Rfree.
RfactorNum. reflection% reflectionSelection details
Rfree0.432 2998 10 %RANDOM
Rwork0.374 ---
obs0.374 29947 99.5 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 57.8921 Å2 / ksol: 0.34846 e/Å3
Displacement parametersBiso mean: 47 Å2
Baniso -1Baniso -2Baniso -3
1-7.17 Å20 Å20 Å2
2--7.17 Å20 Å2
3----14.34 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.75 Å0.56 Å
Luzzati d res low-5 Å
Luzzati sigma a0.27 Å-0.26 Å
Refinement stepCycle: LAST / Resolution: 2.6→16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4014 850 19 93 4976
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.195
X-RAY DIFFRACTIONc_angle_deg2.5
X-RAY DIFFRACTIONc_dihedral_angle_d24.1
X-RAY DIFFRACTIONc_improper_angle_d1.02
LS refinement shellResolution: 2.6→2.76 Å / Rfactor Rfree error: 0.02 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.457 511 10.5 %
Rwork0.369 4359 -
obs--98.7 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2dna-rna-ped.paramdna-rna-ped.top
X-RAY DIFFRACTION3ion.paramion.top
X-RAY DIFFRACTION4water_rep.paramwater.top

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