6CYM

Reversible Covalent Direct Thrombin Inhibitors

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	DIAMOND BEAMLINE I03
Synchrotron site	Diamond
Beamline	I03
Temperature [K]	100
Detector technology	PIXEL
Collection date	2016-12-15
Detector	DECTRIS PILATUS3 6M
Wavelength(s)	0.97625
Spacegroup name	C 2 2 21
Unit cell lengths	91.670, 99.730, 146.010
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	30.000 - 2.900
R-factor	0.18
Rwork	0.176
R-free	0.25400
RMSD bond length	0.010
RMSD bond angle	1.190
Data reduction software	XDS
Data scaling software	Aimless
Phasing software	PHASER
Refinement software	BUSTER (2.11.7)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	30.000	2.950
High resolution limit [Å]	2.900	2.900
Number of reflections	10936
<I/σ(I)>	8.2
Completeness [%]	72.1
Redundancy	6.5

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	BATCH MODE		294	For complex formation, 2 mg human alpha-thrombin (7.1 mg/mL in 50% v/v glycerol) were mixed with 0.5 mM Compound 1 in DMSO and dialyzed overnight into a solution of 20 mM sodium citrate and 1.3 mM Na2SO4 (pH 5.8). After dialysis, another 0.5 mM compound were added and left to incubate at room temperature for 1 h. The protein was centrifuged for 5 min at 4 deg C and 8000x RCF. The pellet, including about 120 uL of buffer, was mixed with 100 uL of a solution containing 50 mM sodium phosphate (pH 7.3), 190 mM sodium chloride, and 0.2 mM Compound 1. The crystal used for data collection was grown from the JCSG+ screen in well A10. The crystal was grown at 20 deg C using a MRC 3-well plate and in a 150 + 150 nL drop with a reservoir of 0.2 M potassium formate and 20% w/v PEG-3350.