3.5 Cryoprotection
of AMPK Crystals
Perform the following steps in a 4C cold room.- Quickly transfer a single crystal into a 2–3μl drop of 1% cryo-
solution; incubate for 10–20 s. - Repeatstep 1, transferring the crystal incrementally into 2%,
3%, 4%, and 5% cryo-solution. Incubate the crystals for
60–120 s in 5% cryo-solution. - After the final incubation, quickly flash cool the crystal in liquid
nitrogen for storage/transport. Typically crystals should dif-
fract to a resolution between 2.5 and 3.5 A ̊.
Fig. 2Full-length phosphorylatedα 2 β 1 γ1 AMPK crystals. Examples of crystals grown in 7–8.5% PEG 3350,
0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl betaine and 0.1 M imidazole (pH 6.2),
co-crystallized with 3AMP, 1A-769662, 1staurosporine, 5S108tide after incubation at 4C for
3 weeks. (a,b) The hexagonal ends of the crystal are clearly visible, (c,d) while from the side the crystals
appear rectangular
24 Christopher G. Langendorf et al.