1–3 days and typically reach full size by 7–10 days. Wells often
become a bit gummy beyond 2 weeks, and crystals beyond this
age may yield poorer diffraction (seeNote 34).Followsteps 6– 9 below for crystal harvesting and X-ray data
collection.- For structural analysis with bound ligands, mix 50μl of soak
solution and 50μl of cryo-solution. Solutions are matched to
the composition of the reservoir in which the crystal is growing
with the addition of 10% (v/v) glycerol (soak solution) or 28%
(v/v) glycerol (cryo-solution). To both cryo- and soak solu-
tion, also add 200μM of staurosporine, 400μM of AMP, and
500 μM of ligand of interest (Fig.3). Centrifuge these samples
for 10 min at 19,000gat room temperature. - For crystal soaking, break film or tape on top of crystal well and
add 0.5μl of soak solution directly onto crystal drop. Reseal
well with tape and incubate overnight at room temperature (see
Note 35). - Harvest the crystals the next day (Fig.4a). Transfer single
crystal by pipette from the soak reservoir well into ~10μlof
fresh soak solution (with compounds) on a glass coverslip
under microscope. Using a mounted loop (seeSubheading
2.3,item 6) attached to a magnetic crystal wand, transfer a
single crystal into 10μl of fresh cryo-solution (with com-
pounds). Allow crystal to soak for at least a few seconds, then
loop again with litholoop and plunge into liquid nitrogen to
freeze (seeNote 36). Transfer to cryo-vial or puck to enable
storage and shipment in cryo-dewars. - Mail dry shipping dewars containing frozen crystals to a syn-
chrotron light source for exposure to X-rays and diffraction
data collection.
For structure solution and analysis, followsteps 10– 13 below.- Process the diffraction images using the software
HKL2000 [33].
Fig. 3Chemical structures of some of the known AMPK direct activators and their prodrugs. AICAR is not a
direct activator by itself but becomes phosphorylated in vivo to generate ZMP which functions as an AMP
mimetic
Biophysical Studies to Evaluate Protein-Ligand Interactions 43