4HDC

Discovery of Selective and Potent Inhibitors of Gram-positive Bacterial Thymidylate Kinase (TMK: Compound 41)

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	ROTATING ANODE
Source details	RIGAKU FR-E+ SUPERBRIGHT
Temperature [K]	140
Detector technology	CCD
Collection date	2009-11-09
Detector	RIGAKU SATURN 944+
Wavelength(s)	1.54
Spacegroup name	P 1 21 1
Unit cell lengths	46.530, 90.736, 48.346
Unit cell angles	90.00, 101.44, 90.00

Refinement procedure

Resolution	23.340 - 2.050
Rwork	0.214
R-free	0.25080
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	In house apo state.
RMSD bond length	0.010
RMSD bond angle	1.110
Data reduction software	XDS
Data scaling software	SCALA
Phasing software	AMoRE
Refinement software	BUSTER

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	40.000	4.410	2.120
High resolution limit [Å]	2.050	3.500	2.050
Rmerge	0.096	0.069	0.339
Number of reflections	23056
<I/σ(I)>	8.9	12	2.4
Completeness [%]	94.5	92.9	98
Redundancy	2.4	2.5	2.3

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6.5	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