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- PDB-6t22: N-terminal domain of EcoKMcrA restriction endonuclease (NEco) in ... -

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Basic information

Entry
Database: PDB / ID: 6t22
TitleN-terminal domain of EcoKMcrA restriction endonuclease (NEco) in complex with T5hmCGA target sequence
Components
  • DNA (5'-D(*GP*AP*AP*TP*(5HC)P*GP*AP*TP*GP*A)-3')
  • DNA (5'-D(*TP*CP*AP*TP*(5HC)P*GP*AP*TP*TP*C)-3')
  • EcoKMcrA modification dependent restriction endonuclease
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 / DNA restriction-modification system / methyl-CpG binding / 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 K-12 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.21 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 30, 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

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Assembly

Deposited unit
A: EcoKMcrA modification dependent restriction endonuclease
B: EcoKMcrA modification dependent restriction endonuclease
C: DNA (5'-D(*TP*CP*AP*TP*(5HC)P*GP*AP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*TP*(5HC)P*GP*AP*TP*GP*A)-3')
E: DNA (5'-D(*TP*CP*AP*TP*(5HC)P*GP*AP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*TP*(5HC)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)47,0336
Polymers47,0336
Non-polymers00
Water7,296405
1
A: EcoKMcrA modification dependent restriction endonuclease
C: DNA (5'-D(*TP*CP*AP*TP*(5HC)P*GP*AP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*TP*(5HC)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,5173
Polymers23,5173
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2900 Å2
ΔGint-18 kcal/mol
Surface area9580 Å2
MethodPISA
2
B: EcoKMcrA modification dependent restriction endonuclease
E: DNA (5'-D(*TP*CP*AP*TP*(5HC)P*GP*AP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*TP*(5HC)P*GP*AP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,5173
Polymers23,5173
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2870 Å2
ΔGint-20 kcal/mol
Surface area9590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)113.417, 113.417, 156.367
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-323-

HOH

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Components

#1: Protein EcoKMcrA modification dependent restriction endonuclease


Mass: 17368.604 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / 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*(5HC)P*GP*AP*TP*TP*C)-3')


Mass: 3025.005 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*(5HC)P*GP*AP*TP*GP*A)-3')


Mass: 3123.080 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 / Water


Mass: 18.015 Da / Num. of mol.: 405 / 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: 3.09 Å3/Da / Density % sol: 60.15 %
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: 0.9195 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 14, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9195 Å / Relative weight: 1
ReflectionResolution: 2.21→33.54 Å / Num. obs: 30298 / % possible obs: 99.2 % / Redundancy: 35.8 % / Biso Wilson estimate: 55.6 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.084 / Rrim(I) all: 0.086 / Net I/σ(I): 38.15
Reflection shellResolution: 2.21→2.34 Å / Redundancy: 20.8 % / Rmerge(I) obs: 1.761 / Mean I/σ(I) obs: 1.91 / Num. unique obs: 4597 / CC1/2: 0.734 / Rrim(I) all: 1.802 / % possible all: 95.4

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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: 6GHC

6ghc


Resolution: 2.21→33.54 Å / Cor.coef. Fo:Fc: 0.974 / Cor.coef. Fo:Fc free: 0.958 / SU B: 8.998 / SU ML: 0.117 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.18 / ESU R Free: 0.163
Details: HYDROGEN ATOMS 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 ...Details: HYDROGEN ATOMS 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
Rfree0.2034 1534 5.1 %RANDOM
Rwork0.157 ---
obs0.1592 28708 99.21 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 158.21 Å2 / Biso mean: 53.522 Å2 / Biso min: 35.08 Å2
Baniso -1Baniso -2Baniso -3
1--0.31 Å2-0.16 Å2-0 Å2
2---0.31 Å20 Å2
3---1.01 Å2
Refinement stepCycle: final / Resolution: 2.21→33.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2320 816 0 405 3541
Biso mean---62.95 -
Num. residues----330
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0050.0133399
X-RAY DIFFRACTIONr_bond_other_d0.0010.0182758
X-RAY DIFFRACTIONr_angle_refined_deg1.211.5484767
X-RAY DIFFRACTIONr_angle_other_deg1.2941.8326424
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2845312
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.34120.759145
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.5415446
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.7731524
X-RAY DIFFRACTIONr_chiral_restr0.0530.2431
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.023348
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02748
LS refinement shellResolution: 2.21→2.26 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.303 97 -
Rwork0.295 1945 -
obs--92.48 %
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)
11.50090.78031.01972.36510.19260.76620.09570.2-0.27330.30410.1513-0.09720.04540.1016-0.2470.05090.041-0.03470.0758-0.02660.1086-16.522829.2994-6.4928
21.48610.9116-0.40081.5722-0.47921.20670.27690.08330.01780.3407-0.1465-0.0809-0.00390.0147-0.13040.13040.0036-0.05310.05310.00510.04615.149851.73715.8043
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A-5 - 145
2X-RAY DIFFRACTION1C1 - 10
3X-RAY DIFFRACTION1D1 - 10
4X-RAY DIFFRACTION2B-5 - 145
5X-RAY DIFFRACTION2E1 - 10
6X-RAY DIFFRACTION2F1 - 10

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