using a peristaltic pump. This may take some time as the sample
is now viscous. Once the entire sample has been applied, attach
the column to the A ̈KTA system and elute off the protein using
100% nickel B buffer at a speed of 1 mL/min and collect 1 mL
factions.- The protein is eluted over a peak covering five 1 mL fractions.
- The protein is now more stable and less sensitive to
temperature. - Now run each fraction on a gel filtration column as described
above, and recheck purity on an SDS-PAGE gel. - Pool the fractions containing AMPK and concentrate to
between 7 and 9 mg/mL using a 30 kDa cutoff membrane
spin concentrator. - Flash freeze the samples in liquid nitrogen and store at 80 C.
3.4 Crystallization
of Full-Length AMPK
α 1 β 1 γ 1
3.4.1 Crystallization
of the Activator Complex
of Phosphorylated Human
AMPK Protein
The procedure that was used to generate the structure in the
publication from the Gamblin lab [4] is described below. Prepare
the AMPK complex stock solution using 7 mg/mL AMPK protein
in 50 mM Tris, pH 8.0, 300 mM NaCl, and 1 mM TCEP, gently
mixed with a threefold molar excess of AMP and onefold molar
excess of staurosporine and 991 compounds (seeNote 7).- Grow crystals by the vapor diffusion technique at 4Cin
hanging drops using the following optimized growth condi-
tions by making the drops in the cold room or on ice. - Prepare drops by mixing equal volumes of AMPK complex
stock solution with well reservoir solution containing 13%
PEG3350, 0.1 M MgCl 2 , 1% glucose, and 0.15% CAPB in
100 mM imidazole (pH 6.2). Typically, fill the reservoirs with
500 μL well solution, and then add 1μL protein solution to a
cover slip, followed rapidly by 1μL of well solution to make
2 μL drops.Quickly place the cover slip on the well to avoid
dehydration of the drop. - Look at the crystals with a microscope at low-power (40) in
the cold room every few days, and record the appearance of the
drops, in terms of precipitate, clear, aggregate, or crystal (also
size). Orthorhombic crystals typically appear in 3–5 days, and
take at least a week to grow to usable sizes, typically 50μmby
100 μm (Fig.2). - For testing X-ray diffraction of the crystal and data collection,
first cryoprotect the crystal. - Transfer crystals into a drop (2μL) of well solution containing
an additional 30% ethylene glycol. Mount the crystal in a
suitably sized mounted cryoloop, then straight away plunge
into liquid nitrogen and store for data collection (Fig.2).
10 Julia A. Hubbard et al.