AMPK Methods and Protocols

3.4.2 Re-Optimization
of Crystallization
Conditions for a New Batch
of Protein

For a new batch of protein, slight re-optimization of growth con-

ditions may be required.

1. Using the protein complex prepared as above, set up a grid of
   about 5 mL each of ten different well solutions. Use between
   10% and 18% PEG3350 (in 2% steps), each at two pH values
   (6.2 and 6.5) while keeping other components constant.


2. Add 500μL of each different well solution to a 24-well crystal-
   lization plate. Set up three crystallization trial drops, for exam-
   ple, 1μL protein plus 1μL well solution, 1.5μL protein plus
   0.5μL well solution, and 0.5μL protein plus 1.5μL well
   solution. Monitor drops daily for the first week for precipita-
   tion and/or crystallization and then every few days
   subsequently.


3. Crystals that grow to at least 50μm in width are best for data
   collection, but several different growth conditions (from the
   grid) and crystals from each drop may need to be tested for
   X-ray diffraction in order to obtain the optimal crystal growth
   conditions. Cryoprotect crystals with well solution with 30%
   ethylene glycol before freezing as before.


4. The crystals can now be tested for X-ray diffraction.

3.4.3 Crystallization
of the Activator Complex
of Unphosphorylated
Human AMPK Protein

The procedure that was used to generate the structures [11] depos-

ited in the PDB from the Gamblin lab is described below.

1. Grow crystals by vapor diffusion at 4C above (Subheading
   3.4.1) but using AMPK protein that has not been subjected to
   in vitro phosphorylation to make the complex with. The best
   condition for crystallization was identified 12% PEG3350,
   300 mM guanidine in 100 mM PIPES buffer at pH 7.2.

Fig. 2Crystallization of AMPK. (a) Example of AMPKα 1 β 1 γ1 crystals growing in a crystallization well. (b)
AMPKα 1 β 1 γ1 mounted in cryoloop

AMPK Crystallisation 11