Structural Studies of mTOR-mLST8 Complexes

Crystals were grown by the hanging-drop vapour diffusion method at 4 °C. Apo-crystals of the mTOR^ΔN–mLST8 complex were grown from 100 mM Tris-Cl, 6-8% (w/v) polyethylene glycol (PEG) 8000, 500 mM NaCl, 10% (v/v) glycerol, 10 mM DTT, pH 8.5. Crystals of the mTOR^ΔN-mLST8 bound to ADP-MgF₃-Mg₂ were grown similarly, except the well-buffer contained 10 mM MgCl₂, 3 mM ADP, and 20 mM NaF. The mTOR^ΔN-mLST8-ATPγS-Mg₂ complex was prepared by soaking apo-crystals for one hour in a stabilization buffer of 50 mM Tris-Cl, pH 8.5, 10 mM Tris-Cl, 8.0, 10% PEG8000, 0.1 M NaCl, 6% glycerol, supplemented with 5 mM MgCl₂ and 1 mM ATPγS. The mTOR^ΔN-mLST8-AMPPNP-Mn₂ complex was prepared by soaking apo-crystals similarly, except the stabilization buffer had a pH of 7.5 and it was supplemented with 1 mM AMPPNP and 2 mM MnCl₂, and the data was collected at the Manganese absorption edge. Crystals of mTOR^ΔN-mLST8-Torin2 and mTOR^ΔN-mLST8-PI-103 were prepared by mixing 1 mM of the inhibitors with the protein. Co-crystals appeared from the same condition as the apo-crystals. Crystals of mTOR^ΔN-mLST8-PP242 were prepared by soaking apo-crystals for 2.5 hours in the stabilization buffer supplemented with 0.2 mM PP242. Apo-crystals were harvested in stabilization buffer, transferred to 50 mM Tris-Cl, pH 8.5, 10 mM Tris-Cl, pH 8.0, 0.1 M NaCl, 14% (w/v) PEG8000, 22% (v/v) glycerol, and were flash-frozen in liquid nitrogen. Crystals with ADP-MgF₃-Mg₂, ATPγS-Mg_2, Torin2, PI103 and PP242 were flash-frozen similarly, except for the presence of the corresponding cofactors or inhibitors (0.1 mM) in the buffers. Diffraction data were collected at -170 °C at the ID24C and ID24E beamlines of the Advanced Photon Source, and they were processed with the HKL suite⁴⁹ .