3B6H
Crystal structure of human prostacyclin synthase in complex with inhibitor minoxidil
X-RAY DIFFRACTION
Starting Model(s)
| Initial Refinement Model(s) | |||
|---|---|---|---|
| Type | Source | Accession Code | Details |
| experimental model | PDB | 2IAG | PDB ENTRY 2IAG |
Crystallization
| Crystalization Experiments | ||||
|---|---|---|---|---|
| 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 |
| Crystal Properties | |
|---|---|
| Matthews coefficient | Solvent content |
| 2.35 | 47.7 |
Crystal Data
| Unit Cell | |
|---|---|
| Length ( Å ) | Angle ( ˚ ) |
| a = 68.762 | α = 90 |
| b = 106.072 | β = 91.81 |
| c = 73.901 | γ = 90 |
| Symmetry | |
|---|---|
| Space Group | P 1 21 1 |
Diffraction
| Diffraction Experiment | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Crystal ID | Scattering Type | Data Collection Temperature | Detector | Detector Type | Details | Collection Date | Monochromator | Protocol | |||||
| 1 | 1 | x-ray | 100 | CCD | ADSC QUANTUM 210 | 2006-09-22 | M | SINGLE WAVELENGTH | ||||||
| Radiation Source | |||||
|---|---|---|---|---|---|
| ID # | Source | Type | Wavelength List | Synchrotron Site | Beamline |
| 1 | SYNCHROTRON | NSRRC BEAMLINE BL13C1 | 0.97622 | NSRRC | BL13C1 |
Data Collection
| Overall | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (Observed) | R Sym I (Observed) | Net I Over Average Sigma (I) | Redundancy | Number Reflections (All) | Number Reflections (Observed) | Observed Criterion Sigma (F) | Observed Criterion Sigma (I) | B (Isotropic) From Wilson Plot | ||||||||
| 1 | 1.62 | 30 | 99.5 | 0.04 | 16.1 | 3.7 | 134319 | 133648 | 1 | 1 | |||||||||
| Highest Resolution Shell | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ID # | Resolution (High) | Resolution (Low) | Percent Possible (All) | Percent Possible (Observed) | R-Sym I (Observed) | Mean I Over Sigma (Observed) | Redundancy | Number Unique Reflections (All) | |||||||||||
| 1.62 | 1.66 | 97.5 | 0.323 | 2 | |||||||||||||||
Refinement
| Statistics | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Diffraction ID | Structure Solution Method | Cross Validation method | Starting model | Resolution (High) | Resolution (Low) | Cut-off Sigma (I) | Cut-off Sigma (F) | Number Reflections (All) | Number Reflections (Observed) | Number Reflections (R-Free) | Percent Reflections (Observed) | R-Factor (All) | R-Factor (Observed) | R-Work | R-Free | R-Free Selection Details | Mean Isotropic B | ||
| X-RAY DIFFRACTION | MOLECULAR REPLACEMENT | THROUGHOUT | PDB ENTRY 2IAG | 1.62 | 29.09 | 1 | 1 | 133648 | 126835 | 6770 | 99.56 | 0.209 | 0.20412 | 0.20284 | 0.22805 | RANDOM | 21.337 | ||
| Temperature Factor Modeling | ||||||
|---|---|---|---|---|---|---|
| Anisotropic B[1][1] | Anisotropic B[1][2] | Anisotropic B[1][3] | Anisotropic B[2][2] | Anisotropic B[2][3] | Anisotropic B[3][3] | |
| RMS Deviations | |
|---|---|
| Key | Refinement Restraint Deviation |
| r_dihedral_angle_2_deg | 30.743 |
| r_dihedral_angle_4_deg | 14.624 |
| r_dihedral_angle_3_deg | 14.306 |
| r_dihedral_angle_1_deg | 5.12 |
| r_scangle_it | 2.201 |
| r_scbond_it | 1.434 |
| r_angle_refined_deg | 1.046 |
| r_mcangle_it | 1.012 |
| r_mcbond_it | 0.599 |
| r_nbtor_refined | 0.304 |
| Non-Hydrogen Atoms Used in Refinement | |
|---|---|
| Non-Hydrogen Atoms | Number |
| Protein Atoms | 7631 |
| Nucleic Acid Atoms | |
| Solvent Atoms | 765 |
| Heterogen Atoms | 156 |
Software
| Software | |
|---|---|
| Software Name | Purpose |
| REFMAC | refinement |
| HKL-2000 | data collection |
| HKL-2000 | data reduction |
| HKL-2000 | data scaling |
| PHASER | phasing |
