4KE6

Crystal structure D196N mutant of Monoglyceride lipase from Bacillus sp. H257 in complex with 1-rac-lauroyl glycerol

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Source details	BRUKER AXS MICROSTAR
Temperature [K]	100
Detector technology	IMAGE PLATE
Collection date	2013-03-22
Detector	MAR scanner 345 mm plate
Wavelength(s)	1.54
Spacegroup name	P 21 21 21
Unit cell lengths	39.185, 182.878, 248.264
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	61.357 - 2.800
R-factor	0.2021
Rwork	0.200
R-free	0.24550
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.008
RMSD bond angle	1.360
Data reduction software	MOSFLM
Data scaling software	SCALA (3.3.21)
Phasing software	PHASER (2.5.2)
Refinement software	PHENIX (1.8_1069)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	73.621	73.621	2.950
High resolution limit [Å]	2.800	8.850	2.800
Rmerge		0.034	0.401
Number of reflections	42482
<I/σ(I)>	9.4	15.8	1.9
Completeness [%]	94.4	89.2	87.6
Redundancy	3.9	4	3.1

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	6.9	293	~0.9 mM of bMGL(D196N) was mixed with 180 mM 1-(rac)-lauroyl glycerol dissolved in 99% EtOH to achieve a final protein-ligand ratio of 1:5. The protein-ligand mixture was incubated at 4 C for 1 hour. Initial crystals were obtained using the Morpheus screen in condition A4. These crystals were used for preparing a seed stock. The optimised crystals were obtained in a drop containing 0.9 mM bMGL(D196N); 56 % v/v MPD, 0.1 M HEPES pH 6.9 and 1:1000 dilution of seed stock in a ratio of 2:2:1 respectively. 1-LG powder was added to these crystallization drops and the crystals were soaked for 8h., VAPOR DIFFUSION, HANGING DROP, temperature 293K