4YZ9
Crystal Structure of human phosphorylated IRE1alpha in complex with a type III kinase inhibitor (GSK2850163A)
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | ROTATING ANODE |
Source details | RIGAKU MICROMAX-007 |
Temperature [K] | 100 |
Detector technology | CCD |
Collection date | 2011-10-04 |
Detector | RAYONIX SX-165mm |
Wavelength(s) | 1.54 |
Spacegroup name | P 21 21 2 |
Unit cell lengths | 244.004, 77.767, 88.313 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 29.182 - 2.463 |
R-factor | 0.2378 |
Rwork | 0.235 |
R-free | 0.28290 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 3p23 |
RMSD bond length | 0.003 |
RMSD bond angle | 0.663 |
Data scaling software | SCALEPACK |
Phasing software | MOLREP |
Refinement software | PHENIX (1.9_1692) |
Data quality characteristics
Overall | Inner shell | Outer shell | |
Low resolution limit [Å] | 29.182 | 30.000 | 2.540 |
High resolution limit [Å] | 2.450 | 5.270 | 2.450 |
Rmerge | 0.073 | 0.028 | 0.592 |
Total number of observations | 377173 | ||
Number of reflections | 59737 | ||
<I/σ(I)> | 9 | 3.1 | |
Completeness [%] | 97.2 | 99.5 | 89.7 |
Redundancy | 6.3 | 6.6 | 6.3 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, SITTING DROP | 7 | 293 | Crystals of pIRE1alpha (547 - 977) with GSK2850163 were prepared by mixing pIRE1alpha with 0.5 mM GSK2850163 and incubating overnight on ice. The crystals were grown at 20 C by vapor diffusion in sitting drops containing 2 ul of protein (13 mg/ml in 50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA , 0.5 mM GSK2850163, 0.25% DMSO ) and 2 ul reservoir solution containing PEG 3350 (16%-22%), 100 mM Hepes pH 7.0, 200 mM Ca2+ acetate. The crystals were thick rods that appeared over 2-5 days and reached full size (0.05 X 0.025 X 0.3 mm) in 2 weeks. Seeding was used to improve crystal quality. The pIRE1alpha + GSK2850163 crystals were frozen in a solution of 20% ethylene glycol, 22% PEG 3350, 0.2 M calcium acetate added in a stepwise manner to the protein drop before mounting the crystal on the loop. |