4YZD

Crystal Structure of human phosphorylated IRE1alpha in complex with ADP-Mg

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	CLSI BEAMLINE 08ID-1
Synchrotron site	CLSI
Beamline	08ID-1
Temperature [K]	100
Detector technology	CCD
Collection date	2011-09-27
Detector	RAYONIX MX-300
Wavelength(s)	0.97949
Spacegroup name	C 1 2 1
Unit cell lengths	191.751, 122.492, 77.876
Unit cell angles	90.00, 107.07, 90.00

Refinement procedure

Resolution	45.643 - 3.102
R-factor	0.2335
Rwork	0.232
R-free	0.27280
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	3p23
RMSD bond length	0.004
RMSD bond angle	0.837
Data scaling software	SCALEPACK
Refinement software	PHENIX (1.9_1692)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	45.643	50.000	3.250
High resolution limit [Å]	3.102	6.760	3.140
Rmerge	0.087	0.052	0.569
Total number of observations	118513
Number of reflections	30914
<I/σ(I)>	9.9
Completeness [%]	100.0	99.9	100
Redundancy	3.8	3.8	3.8

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	6	293	The crystals of pIRE1a with Mg2+-ADP were grown by mixing 2 ul protein solution (10 mg/ml pIRE1a (547-977) in 50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA, 1 mM ADP (100mM ADP stock was ~ pH 7.0), 1 mM MgCl2) with 2 ul reservoir solution (16 % PEG 3350, 200 mM Na+ Malonate pH 6.0) in sitting drops at room temperature. Seeding was used to initiate crystal growth. Crystals appeared the next day and grew to full size in 3 weeks. For data collection, the crystals were frozen in a solution of 20% ethylene glycol, 22% PEG 3350, 200 mM Na+ Malonate pH 6.0 and added to the protein drop before mounting the crystals on the loop.