7ULX

Human DDAH1 soaked with its inhibitor N4-(4-chloropyridin-2-yl)-L-asparagine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 19-BM
Synchrotron site	APS
Beamline	19-BM
Temperature [K]	100
Detector technology	CCD
Collection date	2018-03-12
Detector	ADSC QUANTUM 315r
Wavelength(s)	0.9787
Spacegroup name	P 1 21 1
Unit cell lengths	73.435, 47.590, 81.120
Unit cell angles	90.00, 109.01, 90.00

Refinement procedure

Resolution	28.348 - 1.707
R-factor	0.1934
Rwork	0.191
R-free	0.23510
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3p8e
Data reduction software	iMOSFLM
Data scaling software	HKL-2000
Phasing software	PHENIX
Refinement software	PHENIX (1.14_3260)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	50.000	50.000	1.740
High resolution limit [Å]	1.707	4.640	1.710
Rmerge	0.067	0.046	0.642
Rmeas	0.078	0.055	0.772
Rpim	0.040	0.029	0.420
Number of reflections	56084	2458	2513
<I/σ(I)>	7.9
Completeness [%]	96.9	81.2	88.5
Redundancy	3.7	3.3	2.8
CC(1/2)		0.993	0.624

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		298	Purified DDAH1 was concentrated to approximately 10 mg/mL using an Amicon-Ultra centrifugal filter device (10 kDa MWCO). The protein was crystallized at 25 C using the hanging drop method (Hampton Research, Aliso Viejo, CA) from 25 % (w/v) PEG6000, 0.1 M Tris-HCl, pH 8.2. Further optimization was done manually using a 1:1 well solution : DDAH1 stock solution ratio to improve crystal size and morphology. Crystals grew to their maximum size in 3 weeks and were harvested after approximately 25 days. To determine the structure of DDAH1 in complex with the ligand, the crystals were transferred to a reservoir containing 20 uL of 20 mM of the ligand in the crystallization mother liquor (25% PEG 6000, 0.1 M Tris-HCl, pH 8.2) and soaked for 30 min. Before data collection, crystals with good size and morphology were transferred into a cryoprotection solution (Well Solution supplemented with 25 % (v/v) glycerol) for 1 to 5 seconds using a the cryoloop (Hampton Research, Laguna Niguel, CA). Individual hDDAH1:ligand crystals were flash frozen in liquid nitrogen before use.