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.