- Solve the crystal structure of AMPKxtalby molecular replace-
ment using known structures of different AMPK fragments
using the Phaser application in the CCP4 suite of crystallogra-
phy programs [34]. - Refine the structure using autoBUSTER [35] and Coot [36]
(Fig.4b). - In order to avoid crystallographic bias, it is important to first
refine the “Apo” protein structure and calculate an electron
density map prior to inclusion of the ligand of interest. This
allows interrogation of an “unbiased” difference density map
illustrating regions of mismatch between map and model.
Modeling into this map using ligand-fitting protocols such as
RHOFIT [37] generates an initial docking pose which is sub-
sequently refined to improve the goodness of fit (seeNotes 37
and 38 ) (Fig.4c).
3.4 Enzymatic
Assays
The method below is for a 384-well assay plate; however, volume
adjustments can be made for other plate densities.- Prepare working solution mixtures:Activator mixture—Dilute
compound to 4the desired final compound concentration in
1 kinase buffer. AMPK enzyme mixture—Prepare a 4
AMPK enzyme solution by diluting AMPK in 1kinase buffer
to desired concentration. The selected AMPK concentration
should be optimized so the reaction is linear with good
assay window over a reasonable period of time (seeNote 39).
4 ATP mixture—Prepare ATP solution by diluting ATP in
1 kinase buffer to a concentration equal to 40the reported
Fig. 4X-ray crystallography of AMPKα 1 β 1 γ1 complexes with ligands. (a) Image of crystals of AMPKα 1 β 1 γ 1
(AMPKxtal) growing in sitting drop well. Crystals shown are approximately 50–100μm in size. (b) Overall
topology of AMPKα 1 β 1 γ1 (colored green, cyan, pink) derived from 5KQ5.pdb (10). Activator (PF-06409577) is
bound at the ADaM site and shown in spheres. (c) Electron density is shown (2Fo-Fc map, contoured at 1σ)
with modeled conformation of ligand
44 Ravi G. Kurumbail et al.