5T8I

PI3Kdelta in complex with the inhibitor GS-9901

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ALS BEAMLINE 5.0.1
Synchrotron site	ALS
Beamline	5.0.1
Temperature [K]	100
Detector technology	CCD
Collection date	2013-10-31
Detector	ADSC QUANTUM 315r
Wavelength(s)	1.00
Spacegroup name	C 2 2 21
Unit cell lengths	64.045, 142.197, 222.590
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	45.880 - 2.600
R-factor	0.256
Rwork	0.252
R-free	0.31250
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.003
RMSD bond angle	0.703
Data reduction software	HKL-2000
Data scaling software	SCALEPACK
Phasing software	EPMR
Refinement software	PHENIX (1.9_1692)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	2.640
High resolution limit [Å]	2.600	2.600
Number of reflections	28814
<I/σ(I)>	25.6	1.8
Completeness [%]	91.4	89.8
Redundancy	5.6	5.1
CC(1/2)		0.646

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION		293	Crystals used for seeding were obtained by vapor diffusion at 20 degrees C with 25 nl of 11 mg/ml (delta)ABD-p110(delta) and 0.9 mM of a p110(delta) inhibitor in protein storage buffer (20 mMTris, pH 7.2, 50 mM (NH4)2SO4, 1% v/v ethylene glycol, 1% (w/v) betaine, 0.3 microM CHAPS, and 5 mM TCEP) added to 25 nl of reservoir solution (25% (w/v) PEG 3350, 0.1 M bis-tris (pH 6.5). Crystals were pulverized, pooled together in the reservoir solution, vortexed with a Seed-Bead (Hampton Research) for 45s, flash-frozen in liquid nitrogen, and stored at -80 degrees C. For seeding crystallization trials, the frozen seed was thawed and diluted 500-fold in 25% (v/v) PEG 300, 0.1 M Tris (pH 8.5). Preparation of diffraction quality (delta)ABD-p110(delta):GS-9901 crystals started with a mixture of 0.48 microL of 2.5% (w/v) n-dodecyl-(beta)-D-maltoside, 0.30 microL of 20 mM ligand, and 12 microL of 12 mg/ml (delta)ABD-p110(delta) in storage buffer (described above). The mixture was allowed to sit for 1 h at room temperature instead of 4 degrees C to prevent the formation of white precipitate. Seeded vapor diffusion droplets were assembled by adding 90 nl of the 500-fold diluted seed (described above) to 100 nl of the n-dodecyl-(beta)-D-maltoside-ligand-(delta)ABD-p110(delta) mixture. The droplets were equilibrated against reservoir wells containing 50 microL of 1% to 30% (v/v) PEG 300, 0.1 M Tris (pH 8.5) at 20 degrees C. Crystals appeared in 2-5 days across the 1% to 30% PEG range. Crystals were cryoprotected in 20% (w/v) glycerol, 25% (w/v) PEG 300, 0.1 M Tris (pH 8.5), 50 mM ammonium sulfate, 0.2% (w/v) n-dodecyl-(beta)-D-maltoside, 0.2 mM ligand and were flash-frozen in liquid nitrogen for data collection.