4YZC

Crystal structure of pIRE1alpha in complex with staurosporine

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	APS BEAMLINE 21-ID-D
Synchrotron site	APS
Beamline	21-ID-D
Temperature [K]	100
Detector technology	CCD
Collection date	2012-02-17
Detector	ADSC QUANTUM 315
Wavelength(s)	0.97872
Spacegroup name	P 21 21 21
Unit cell lengths	49.337, 155.260, 155.643
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	40.225 - 2.494
R-factor	0.2489
Rwork	0.247
R-free	0.28920
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3p23
RMSD bond length	0.003
RMSD bond angle	0.648
Data scaling software	SCALEPACK
Phasing software	MOLREP
Refinement software	PHENIX (1.9_1692)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	40.230	2.583
High resolution limit [Å]	2.494	2.494
Rmerge	0.097	0.134
Total number of observations	226940
Number of reflections	41927
<I/σ(I)>	13.36
Completeness [%]	98.2	88.06
Redundancy	6.5	6.2

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	293	The complex of pIRE1a (547-977) with staurosporine was prepared by mixing the protein with 0.5 mM staurosporine and incubated overnight on ice. The crystals were grown at 20C by vapor diffusion in sitting drop containing 2 ul of protein (13 mg/ml in 50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA) and 2ul reservoir solution containing PEG 300 (30%-40%), 100 mM Hepes pH 7.5, 200 mM KCl. Small hexagonal plates appeared over 5-10 days and reached full size (0.05 X 0.75 X 0.15 mm) in ~20 days. The crystals were flash-frozen in liquid N2 directly from the crystallization drop. All diffraction data was collected at the Advanced Photon Source, Argonne National Laboratories, Life Sciences CAT, Sector 21