- For a new batch of protein of unphosphorylated protein, the
optimal condition for crystallization is likely to have also
changed slightly so as set up a grid of different well solutions
with a range of different PEG3350 concentrations and
pH. Typically this would be 10–16% PEG3350, pH 7.0, 7.2,
and 7.4. Also try varying the composition of the drops by
changing the protein to well ratios; similarly try 1:1, 1.5:0.5,
and 0.5:1.5. - Monitor the appearance of the drops as before using a micro-
scope in the cold room. Crystals typically appear after around
3–10 days. - Cryoprotect by moving crystals into well solution with an
additional 30% ethylene glycol, before plunging into liquid
nitrogen and testing for diffraction of X-rays.
3.5 X-Ray Data
Collection, Processing,
and Model Building
of AMPK
The instruments and procedures for collecting and processing
X-ray data will vary dependent upon the access to and expertise of
the individual scientist. For the scientist new to X-ray crystallogra-
phy, this process requires specialist training and is outside the scope
of this chapter.
Briefly, in our hands diffraction data were collected at 100K on
a Pilatus 2 M detector (Dectris), Diamond Light Source, Oxford.
The phosphorylated full-length AMPK activator complex crystals
grown under these conditions were found to belong to the space
group P2 1. For the unphosphorylated AMPK complex, the crystals
belonged to the P1 2 1 1 space group.
Data were integrated using the program DENZO and scaled
with SCALEPACK. The structure was solved by molecular replace-
ment using PHASER, and standard refinement was carried out with
PHENIX using 2Y94.pdb and 2F15.pdb as the search models, with
manual model building with COOT. General crystallographic cal-
culations were carried out using the CCP4 package. Figures were
created with PyMOL.4 Notes
- The pH of Tris is temperature sensitive. Cool water in cold
room before preparation of lysis buffer. Use buffer directly
from cold room. - While this centrifugation step is ongoing, this is an opportunity
to prepare the nickel column for the purification later in day 2. - Overnight the AMPK protein remains attached to the column
at 4C in the cold room. Never elute the AMPK protein off the
nickel column and leave overnight in imidazole as it is highly
prone to aggregation at this stage.
12 Julia A. Hubbard et al.