6R64

N-terminal domain of modification dependent EcoKMcrA restriction endonuclease (NEco) in complex with C5mCGG target sequence

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	BESSY BEAMLINE 14.1
Synchrotron site	BESSY
Beamline	14.1
Temperature [K]	100
Detector technology	PIXEL
Collection date	2018-07-10
Detector	DECTRIS PILATUS 6M
Wavelength(s)	0.91840
Spacegroup name	P 61 2 2
Unit cell lengths	112.432, 112.432, 155.457
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	45.550 - 2.640
R-factor	0.19237
Rwork	0.191
R-free	0.22921
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6ghc
RMSD bond length	0.006
RMSD bond angle	1.146
Data reduction software	XDS
Data scaling software	XDS
Phasing software	PHASER
Refinement software	REFMAC (5.8.0189)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	45.550	2.800
High resolution limit [Å]	2.640	2.640
Rmeas	0.230	1.509
Number of reflections	17671	2761
<I/σ(I)>	12.52	1.97
Completeness [%]	99.8	99.2
Redundancy	16	16.7
CC(1/2)	0.997	0.764

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	6.5	294	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.