7ULU

Human DDAH1 soaked with its inhibitor ClPyrAA

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	2019-06-15
Detector	DECTRIS EIGER X 9M
Wavelength(s)	0.9787
Spacegroup name	P 1 21 1
Unit cell lengths	73.810, 46.850, 80.270
Unit cell angles	90.00, 109.15, 90.00

Refinement procedure

Resolution	62.560 - 2.200
R-factor	0.2103
Rwork	0.207
R-free	0.26680
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3p8e
Data reduction software	MOSFLM
Data scaling software	Aimless (0.7.4)
Phasing software	PHENIX
Refinement software	PHENIX (1.19.2_4158)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	75.830	75.830	2.270
High resolution limit [Å]	2.200	9.070	2.200
Rmerge	0.077	0.044	1.084
Rmeas	0.086	0.050
Rpim	0.037	0.023	0.699
Total number of observations	123773
Number of reflections	24478	417	1348
<I/σ(I)>	6.8
Completeness [%]	91.8	99.2	59.8
Redundancy	5.1	4.9	3.2
CC(1/2)	0.995	0.987	0.383

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP		293	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 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.