4HEJ | pdb_00004hej

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

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
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

Crystal Properties
Matthews coefficient	Solvent content
2.15	42.73

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 45.348	α = 90
b = 90.308	β = 100.69
c = 50.07	γ = 90

Symmetry
Space Group	P 1 21 1

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	140	CCD	RIGAKU SATURN 944+	microfocus rotating anode with 70 uM focal spot	2008-08-29	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	ROTATING ANODE	RIGAKU FR-E+ SUPERBRIGHT	1.54

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	2.1	25.68	76.7	0.075	11.4	3.38	26805	20571	1	1.3	28.73

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	2	2.07	23.5		0.147	3.3	1.75	2669

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Resolution (High)	Resolution (Low)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work (Depositor)	R-Work (DCC)	R-Free (Depositor)	R-Free (DCC)	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	2	25.68	19634	987	73.24	0.195	0.1929	0.19	0.2347	0.22	RANDOM, 5%	30.16

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]
0.0116		-4.3585	2.7813		-2.7929

RMS Deviations
Key	Refinement Restraint Deviation
t_other_torsion	18.26
t_omega_torsion	3.01
t_angle_deg	1.15
t_bond_d	0.01
t_dihedral_angle_d
t_trig_c_planes
t_gen_planes
t_it
t_improper_torsion
t_chiral_improper_torsion

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3147
Nucleic Acid Atoms
Solvent Atoms	193
Heterogen Atoms	33

Software

Software
Software Name	Purpose
JDirector	data collection
AMoRE	phasing
BUSTER	refinement
d*TREK	data reduction
d*TREK	data scaling

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM157729.