Crystallization of PI3K p110β-p85β Complex

Mouse His₆-p110β(1-1064)/p85β-icSH2(423-722) complex was diluted to 4 mg/ml, mixed with 20 mM (final concentration) sodium phenyl phosphate (Sigma P-7751) and 150 μM of the PI3K inhibitor GDC0941 (Folkes et al., 2008 (link)). The initial crystallization conditions were obtained from a broad screen of 1056 conditions (Stock et al., 2005 (link)) in 96-well MRC crystallization plates (SWISSCI AG, Zug, Switzerland). Additives (GDC0941 and phenyl phosphate) were identified by differential scanning fluorimetry (see Supplemental Experimental Procedures). Optimal crystals were obtained at 22°C in hanging drops over reservoirs of 24-well plates (Hampton Research, Aliso Viejo, CA) containing 12% polyethylene glycol 3350, 0.1 M potassium citrate at pH 6, and 0.4 M lithium sulfate. The drops contained 1 μl each of protein and reservoir solutions. The crystals were cryoprotected by stepwise addition of cryoprotectants consisting of the reservoir solution with 20 mM sodium phenyl phosphate, 150 μM of GDC0941, and an increasing concentration of glycerol up to 20% (in 5% increments). Crystals were flash frozen in liquid nitrogen.