Computational protocol: Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells

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Protocol publication

[…] Full-length AMPK, His-α2 (human 1–552), β1 (human 1–270) and γ1 (human, 1–331) were expressed in E. coli and purified using nickel affinity chromatrography and gel filtration as previously described []. A stock solution was prepared at 5 mg/ml in 50 mM Tris (pH 8.0), 300 mM NaCl and 1 mM tris(2-carboxyethyl)phosphine, mixed with a 4-fold molar excess of AMP and 1-fold of staurosporine and 991 compound. Crystals were grown by the vapour diffusion technique at 4°C in hanging drops. Drops were prepared by mixing equal volumes of protein complex with reservoir solution containing 12% polyethylene glycol (PEG3350), 300 mM guanidine in 100 mM piperazine-N,N′-bis(2-ethanesulfonic acid) buffer (pH 7.2). Crystals were first transferred into mother liquid with an additional 25% ethylene glycol, before plunging into liquid nitrogen. Diffraction data were collected on a Pilatus 2 M detector (Dectris), Diamond Lightsource, Oxford. Data were integrated using Denzo and scaled with Scalepack. The structure was solved by molecular replacement using Phaser and standard refinement was carried out with Phenix using 4CFE.pdb as search models, with a manual model building with COOT []. General crystallographic calculations were carried out using the CCP4 package []. Figures were created with Pymol (http://pymol.sourceforge.net/). […]

Pipeline specifications

Software tools PHENIX, Coot, CCP4, PyMOL
Application Protein structure analysis
Organisms Homo sapiens
Chemicals Ampicillin, Deoxyglucose