Crystallization of WDR5-PTEN Complexes

For the crystals of WDR5^22–334-PTENα-NTE^1–173, purified WDR5^22–334 was mixed with PTENα-NTE^1–173 at a molar ratio of 1:1 and with trypsin at a mass ratio of 1:1000 (trypsin: protein mixture) [54 (link), 55 (link)]. Then, the mixture was crystallized using the sitting-drop vapor diffusion method at 18 °C by adding 0.5 μL of the protein mixture (8 mg/mL) with 0.5 μL of the reservoir solution. The complex of WDR5^22–334-PTENα-NTE^1–173 crystallized in a buffer containing 0.2 M lithium sulfate monohydrate, 0.1 M HEPES, pH 7.5, and 25% w/v polyethylene glycol 3,350. As for the crystals of WDR5^1–334, purified WDR5^1–334 protein was mixed with PTENα-NTE^116–148 peptide at a molar ratio of 1:3, and crystallized in a buffer containing 0.1 M magnesium formate dihydrate and 15% w/v polyethylene glycol 3,350. Before flash-freezing crystals in liquid nitrogen, crystals were soaked in a cryoprotectant consisting of 85% reservoir solution and 15% glycerol.