JNK3 protein (Ser40–Glu402) was prepared as described in (Xie et al., 1998, Structure 6(8), 983-991).

Crystals were first grown, via hanging-drop vapor diffusion, with 1 mM AMP-PNP and 2 mM MgCl2 added to the protein. One microliter of protein was combined with one microliter of well solution (18-24 % (v/v) polyethylene glycol monomethyl ether (average Mr = 550), 10 % (v/v) ethylene glycol, 20 mM 2-mercaptoethanol, and 100 mM HEPES at pH 7.5).

For small-molecule inhibitor structures, the above crystals were crushed and used to seed crystallization trials (conditions as above) where the protein solution contained 0.5 mM inhibitor and 5 % (v/v) DMSO. Before data collection, crystals were equilibrated in their reservoir solution for 2–5 min before being flash-frozen in nitrogen gas for X-ray data collection at -170°C.

Please note that the crystal structure is UNACTIVATED Jnk3 (as opposed to the binding experiment), so the flexible loop is almost certainly in a different conformation.