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. |