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- PDB-6t21: N-terminal domain of EcoKMcrA restriction endonuclease (NEco) in ... -

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Basic information

Entry
Database: PDB / ID: 6t21
TitleN-terminal domain of EcoKMcrA restriction endonuclease (NEco) in complex with T5mCGA target sequence
Components
  • 5-methylcytosine-specific restriction enzyme A
  • DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')
  • DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')
KeywordsHYDROLASE / EcoKMcrA / NEco / N-TERMINAL DOMAIN / MODIFICATION DEPENDENT RESTRICTION / 5-METHYLCYTOSINE / 5MC / 5-HYDROXYMETHYLCYTOSINE / 5HMC / HNH ENDONUCLEASE / BBA-ME NUCLEASE
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / methyl-CpG binding / DNA restriction-modification system / endonuclease activity / zinc ion binding
Similarity search - Function
: / HNH endonuclease / HNH endonuclease / HNH nucleases / HNH nuclease
Similarity search - Domain/homology
DNA / Type IV methyl-directed restriction enzyme EcoKMcrA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.07 Å
AuthorsSlyvka, A. / Zagorskaite, E. / Czapinska, H. / Sasnauskas, G. / Bochtler, M.
Citation
Journal: Nucleic Acids Res. / Year: 2019
Title: Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping.
Authors: Slyvka, A. / Zagorskaite, E. / Czapinska, H. / Sasnauskas, G. / Bochtler, M.
History
DepositionOct 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 23, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Database references / Category: citation
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.title / _citation.year
Revision 1.2Dec 18, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 5-methylcytosine-specific restriction enzyme A
B: 5-methylcytosine-specific restriction enzyme A
C: DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')
E: DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)46,9696
Polymers46,9696
Non-polymers00
Water7,764431
1
A: 5-methylcytosine-specific restriction enzyme A
C: DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,4853
Polymers23,4853
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4140 Å2
ΔGint-8 kcal/mol
Surface area9200 Å2
2
B: 5-methylcytosine-specific restriction enzyme A
E: DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,4853
Polymers23,4853
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4110 Å2
ΔGint-8 kcal/mol
Surface area9210 Å2
Unit cell
Length a, b, c (Å)112.882, 112.882, 155.751
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
Components on special symmetry positions
IDModelComponents
11B-340-

HOH

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Components

#1: Protein 5-methylcytosine-specific restriction enzyme A / EcoKMcrA


Mass: 17368.604 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mcrA, rglA, b1159, JW1145 / Plasmid: PET15BM / Production host: Escherichia coli (E. coli)
References: UniProt: P24200, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: DNA chain DNA (5'-D(*TP*CP*AP*TP*(5CM)P*GP*AP*TP*TP*C)-3')


Mass: 3009.006 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: DNA chain DNA (5'-D(*GP*AP*AP*TP*(5CM)P*GP*AP*TP*GP*A)-3')


Mass: 3107.081 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 431 / Source method: isolated from a natural source / Formula: H2O
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: 3.05 Å3/Da / Density % sol: 59.66 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 1.8 ul of protein-DNA solution (436:523 uM) was mixed with 2.2 ul of the F1 condition of the PACT premier crystal screen (MDL) (0.2 M Sodium fluoride, 0.1 M Bis-Tris propane, pH 6.5, 20% PEG ...Details: 1.8 ul of protein-DNA solution (436:523 uM) was mixed with 2.2 ul of the F1 condition of the PACT premier crystal screen (MDL) (0.2 M Sodium fluoride, 0.1 M Bis-Tris propane, pH 6.5, 20% PEG 3350) and cryo-protected with an addition of 25% glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9793 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 13, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.07→33.38 Å / Num. obs: 36261 / % possible obs: 99.5 % / Redundancy: 38.87 % / Biso Wilson estimate: 53 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.103 / Rrim(I) all: 0.105 / Net I/σ(I): 30.17
Reflection shellResolution: 2.07→2.19 Å / Redundancy: 38.92 % / Rmerge(I) obs: 2.299 / Mean I/σ(I) obs: 1.98 / Num. unique obs: 5681 / CC1/2: 0.758 / Rrim(I) all: 2.329 / % possible all: 98.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0253refinement
XDSdata reduction
XDSdata scaling
MOLREPphasing
ARP/wARPmodel building
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6R64

6r64


Resolution: 2.07→33.38 Å / Cor.coef. Fo:Fc: 0.976 / Cor.coef. Fo:Fc free: 0.961 / SU B: 7.669 / SU ML: 0.103 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.148 / ESU R Free: 0.141
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. TLS REFINEMENT HAS BEEN USED. U VALUES : WITH TLS ADDED. THE CLUSTERS OF SOLVENT MOLECULES BETWEEN RESIDUES 46 AND 94 AND NEXT TO RESIDUE ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. TLS REFINEMENT HAS BEEN USED. U VALUES : WITH TLS ADDED. THE CLUSTERS OF SOLVENT MOLECULES BETWEEN RESIDUES 46 AND 94 AND NEXT TO RESIDUE 54 OF CHAIN B LIKELY CORRESPOND TO DISORDERED GLYCEROL MOLECULES. THE ELECTRON DENSITY IS NOT DEFINED ENOUGH TO UNAMBIGUOUSLY MODEL THEM.
RfactorNum. reflection% reflectionSelection details
Rfree0.2013 1824 5.1 %RANDOM
Rwork0.159 ---
obs0.161 34065 98.62 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 135.27 Å2 / Biso mean: 52.793 Å2 / Biso min: 33.27 Å2
Baniso -1Baniso -2Baniso -3
1--0.3 Å2-0.15 Å2-0 Å2
2---0.3 Å20 Å2
3---0.99 Å2
Refinement stepCycle: final / Resolution: 2.07→33.38 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2320 812 0 431 3563
Biso mean---64.84 -
Num. residues----330
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0173461
X-RAY DIFFRACTIONr_bond_other_d0.0010.022818
X-RAY DIFFRACTIONr_angle_refined_deg1.1521.7684853
X-RAY DIFFRACTIONr_angle_other_deg0.9236567
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.1975326
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.06122.96125
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.63815461
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.5521525
X-RAY DIFFRACTIONr_chiral_restr0.0660.2476
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.023439
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02758
LS refinement shellResolution: 2.07→2.12 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.354 116 -
Rwork0.314 2124 -
obs--84.95 %
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)
11.77910.95190.92131.71350.34970.49660.1590.1771-0.38970.2260.0657-0.10150.06370.0925-0.22470.05030.0239-0.02360.0319-0.05040.1062-16.079628.8253-6.3212
21.54290.9161-0.31051.32010.04710.63440.1845-0.0261-0.06590.3153-0.1107-0.10340.0461-0.0056-0.07370.1174-0.0019-0.04110.0286-0.00180.02145.412751.52665.9053
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A-1 - 143
2X-RAY DIFFRACTION1C1 - 10
3X-RAY DIFFRACTION1D1 - 10
4X-RAY DIFFRACTION2B-1 - 143
5X-RAY DIFFRACTION2E1 - 10
6X-RAY DIFFRACTION2F1 - 10

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