7ULV

Human DDAH1 soaked with its inactivator S-((4-chloropyridin-2-yl)methyl)-L-cysteine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 21-ID-D
Synchrotron site	APS
Beamline	21-ID-D
Temperature [K]	100
Detector technology	PIXEL
Collection date	2018-11-19
Detector	DECTRIS EIGER X 9M
Wavelength(s)	0.97872
Spacegroup name	P 1 21 1
Unit cell lengths	46.404, 220.991, 73.645
Unit cell angles	90.00, 91.08, 90.00

Refinement procedure

Resolution	73.630 - 2.370
R-factor	0.2418
Rwork	0.237
R-free	0.26920
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3p8e
Data reduction software	xia2
Data scaling software	xia2
Phasing software	PHENIX
Refinement software	PHENIX (1.19.2_4158)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	73.660	73.700	2.410
High resolution limit [Å]	2.370	6.420	2.370
Rmeas	0.128	0.073	0.836
Rpim	0.062	0.036	0.406
Total number of observations	256173	12756	12131
Number of reflections	59571	3057	2820
<I/σ(I)>	8	13	3.5
Completeness [%]	99.1	100	94.1
Redundancy	4.3	4.2	4.3

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 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.