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- PDB-6r64: N-terminal domain of modification dependent EcoKMcrA restriction ... -

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Basic information

Entry
Database: PDB / ID: 6r64
TitleN-terminal domain of modification dependent EcoKMcrA restriction endonuclease (NEco) in complex with C5mCGG target sequence
Components
  • 5-methylcytosine-specific restriction enzyme A
  • DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')
  • DNA (5'-D(*TP*CP*AP*CP*(5CM)P*GP*GP*TP*TP*C)-3')
KeywordsHYDROLASE / EcoKMcrA / NEco / N-TERMINAL DOMAIN / MODIFICATION DEPENDENT RESTRICTION / 5-METHYLCYTOSINE / 5MC / 5-HYDROXYMETHYLCYTOSINE / 5HMC / HNH ENDONUCLEASE / BBA-ME NUCLEASE / ScoMcrA
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 K12 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.64 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
DepositionMar 26, 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*CP*(5CM)P*GP*GP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')
E: DNA (5'-D(*TP*CP*AP*CP*(5CM)P*GP*GP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)46,9736
Polymers46,9736
Non-polymers00
Water2,630146
1
A: 5-methylcytosine-specific restriction enzyme A
C: DNA (5'-D(*TP*CP*AP*CP*(5CM)P*GP*GP*TP*TP*C)-3')
D: DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,4873
Polymers23,4873
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: 5-methylcytosine-specific restriction enzyme A
E: DNA (5'-D(*TP*CP*AP*CP*(5CM)P*GP*GP*TP*TP*C)-3')
F: DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)23,4873
Polymers23,4873
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)112.432, 112.432, 155.457
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122

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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 K12 (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*CP*(5CM)P*GP*GP*TP*TP*C)-3')


Mass: 3009.994 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*GP*AP*AP*CP*(5CM)P*GP*GP*TP*GP*A)-3')


Mass: 3108.069 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 146 / Source method: isolated from a natural source / Formula: H2O

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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: 8.29 Å3/Da / Density % sol: 85.17 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: NEco in the crystallization buffer (20 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 2 mM DTT) was concentrated to 8.7 mg/ml and mixed in the 1:1.2 ratio with 10-mer TCAC5mCGGTTC ...Details: NEco in the crystallization buffer (20 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 2 mM DTT) was concentrated to 8.7 mg/ml and mixed in the 1:1.2 ratio with 10-mer TCAC5mCGGTTC oligonucleotide, annealed to its complementary GAAC5mCGTGA strand. Crystals were grown by mixing 1.8 ul of the protein-DNA mixture with 2.2 ul of the condition F1 of the PACT premier crystal screen (MDL) (0.2 M NaF, 0.1 M Bis-Tris propane, pH 6.5, 20% PEG3350). Crystals were cryo-protected by the addition of 25% glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.9184 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 10, 2018
RadiationMonochromator: KMC-1 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 2.64→45.55 Å / Num. obs: 17671 / % possible obs: 99.8 % / Redundancy: 16 % / Biso Wilson estimate: 50.9 Å2 / CC1/2: 0.997 / Rrim(I) all: 0.23 / Rsym value: 0.223 / Net I/σ(I): 12.52
Reflection shellResolution: 2.64→2.8 Å / Redundancy: 16.7 % / Mean I/σ(I) obs: 1.97 / Num. unique obs: 2761 / CC1/2: 0.764 / Rrim(I) all: 1.509 / Rsym value: 1.463 / % possible all: 99.2

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Processing

Software
NameVersionClassification
REFMAC5.8.0189refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
ARP/wARPmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 6GHC

6ghc


Resolution: 2.64→45.55 Å / Cor.coef. Fo:Fc: 0.953 / Cor.coef. Fo:Fc free: 0.926 / SU B: 11.193 / SU ML: 0.219 / Cross valid method: THROUGHOUT / ESU R: 0.492 / ESU R Free: 0.273 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.22921 884 5 %RANDOM
Rwork0.19052 ---
obs0.19237 16786 99.82 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 53.738 Å2
Baniso -1Baniso -2Baniso -3
1--1.73 Å2-0.87 Å2-0 Å2
2---1.73 Å20 Å2
3---5.62 Å2
Refinement stepCycle: LAST / Resolution: 2.64→45.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2320 812 5 141 3278
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0173305
X-RAY DIFFRACTIONr_bond_other_d0.0020.022656
X-RAY DIFFRACTIONr_angle_refined_deg1.1461.7524632
X-RAY DIFFRACTIONr_angle_other_deg1.04836169
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.385294
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.59223.109119
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.42415417
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.8981522
X-RAY DIFFRACTIONr_chiral_restr0.0630.2459
X-RAY DIFFRACTIONr_gen_planes_refined0.0030.023160
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02702
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.3064.7141173
X-RAY DIFFRACTIONr_mcbond_other1.3064.7141172
X-RAY DIFFRACTIONr_mcangle_it2.2587.0671468
X-RAY DIFFRACTIONr_mcangle_other2.2577.0671469
X-RAY DIFFRACTIONr_scbond_it1.8946.2132132
X-RAY DIFFRACTIONr_scbond_other1.8946.2172133
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.2999.3463165
X-RAY DIFFRACTIONr_long_range_B_refined4.94358.9273919
X-RAY DIFFRACTIONr_long_range_B_other4.91558.8593889
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.639→2.708 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.358 63 -
Rwork0.3 1193 -
obs--98.2 %

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