4HEJ

Discovery of Selective and Potent Inhibitors of Gram-positive Bacterial Thymidylate Kinase (TMK): Compund 16

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Source details	RIGAKU FR-E+ SUPERBRIGHT
Temperature [K]	140
Detector technology	CCD
Collection date	2008-08-29
Detector	RIGAKU SATURN 944+
Wavelength(s)	1.54
Spacegroup name	P 1 21 1
Unit cell lengths	45.348, 90.308, 50.070
Unit cell angles	90.00, 100.69, 90.00

Refinement procedure

Resolution	25.680 - 2.000
R-factor	0.195
Rwork	0.193
R-free	0.23470
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.010
RMSD bond angle	1.150
Data reduction software	d*TREK
Data scaling software	d*TREK
Phasing software	AMoRE
Refinement software	BUSTER (2.11.2)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	25.680	2.710	2.070
High resolution limit [Å]	2.100	2.520	2.000
Rmerge	0.075	0.079	0.147
Number of reflections	20571
<I/σ(I)>	11.4	8.1	3.3
Completeness [%]	76.7	98	23.5
Redundancy	3.38	3.74	1.75

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6.8	293	To obtain the inhibitor bound crystal form of TMK-S.aureus crystals were initially grown in the absence of compound using the sitting drop method at 20 C with a reservoir solution of 100 mM PCPT (propionate-cacodylate-bistris propane buffer) pH 7-8, 21-24% PEG 3350, 200 mM Mg2Cl using 1:1 protein:reservoir solution with the protein solution at 13 mg/mL. Crystals were harvested and soaked overnight in a solution containing 100 mM PCPT, 35% PEG 3350, 200 mM Mg2Cl and 1-2 mM TK-666 from a 100 mM DMSO stock. After soaking the crystals were cryoprotected by soaking for 15 minutes in compound-soak solution supplemented with 20% ethylene glycol. Cryoprotected crystals were mounted on nylon loops and flash-cooled in liquid nitrogen., VAPOR DIFFUSION, SITTING DROP, temperature 293K