3B6H
Crystal structure of human prostacyclin synthase in complex with inhibitor minoxidil
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | NSRRC BEAMLINE BL13C1 |
| Synchrotron site | NSRRC |
| Beamline | BL13C1 |
| Temperature [K] | 100 |
| Detector technology | CCD |
| Collection date | 2006-09-22 |
| Detector | ADSC QUANTUM 210 |
| Wavelength(s) | 0.97622 |
| Spacegroup name | P 1 21 1 |
| Unit cell lengths | 68.762, 106.072, 73.901 |
| Unit cell angles | 90.00, 91.81, 90.00 |
Refinement procedure
| Resolution | 29.090 - 1.620 |
| R-factor | 0.20412 |
| Rwork | 0.203 |
| R-free | 0.22805 |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | 2iag |
| RMSD bond length | 0.007 |
| RMSD bond angle | 1.046 |
| Data reduction software | HKL-2000 |
| Data scaling software | HKL-2000 |
| Phasing software | PHASER |
| Refinement software | REFMAC (5.2.0019) |
Data quality characteristics
| Overall | Outer shell | |
| Low resolution limit [Å] | 30.000 | 1.660 |
| High resolution limit [Å] | 1.620 | 1.620 |
| Number of reflections | 133648 | |
| <I/σ(I)> | 16.1 | 2 |
| Completeness [%] | 99.5 | 97.5 |
| Redundancy | 3.7 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 8 | 277 | Crystallization of native human prostacyclin synthase (hPGIS). The native recombinant PGIS (25 mg/ml in the gel filtration buffer containing 20 mM Tris, pH 8.0 and 150 mM NaCl) was found to crystallize spontaneously into needle clusters in the Eppendorf tube when stored at 4 C for about a week. A combination of microseeding technique and the hanging-drop vapor-diffusion method was then used to improve crystal quality. Specifically, seed stock was prepared by crushing these initial PGIS needle clusters with the Seed Bead Kit (Hampton Research). Following four 10-fold serial dilutions of the seed stock using the gel filtration buffer, 1 microliter of diluted seeds was added into a 10 microliter drop of freshly concentrated PGIS sample, and equilibrated at 4 C against 450 microliter of the gel filtration buffer. Crystallization of hPGIS-minoxidil complex: The inhibitor-enzyme complex was prepared by adding minoxidil (final concentration 0.1 mM) to a 20 mg/ml hPGIS stock solution. Crystals of the complex were then obtained by co-crystallization, with the technique described above using ligand-free hPGIS microcrystals as seeds., VAPOR DIFFUSION, HANGING DROP, temperature 277K |
