To purify the GM64–237:PP1α7–330 complex for NMR spectroscopy analysis, PP1α7–330 was lysed in PP1 Lysis Buffer [25 mM tris (pH 8.0), 700 mM NaCl, 5 mM imidazole, 1 mM MnCl2, and 0.1% Triton X-100], clarified by ultracentrifugation, and immobilized on Ni2+-NTA resin. Bound His6-PP1 was washed with PP1 Buffer A [25 mM tris (pH 8.0), 700 mM NaCl, 5 mM imidazole, and 1 mM MnCl2], followed by a stringent wash containing 6% PP1 Buffer B [25 mM tris (pH 8.0), 700 mM NaCl, 250 mM imidazole, and 1 mM MnCl2] at 4°C. The protein was eluted using PP1 Buffer B and purified using SEC [Superdex 200 26/60 (GE Healthcare)] pre-equilibrated in ITC Buffer [20 mM tris (pH 8), 500 mM NaCl, 0.5 mM TCEP, and 1 mM MnCl2]. Peak fractions were incubated overnight with TEV protease at 4°C. The cleaved protein was incubated with Ni2+-NTA beads (GE Healthcare), and the flow-through was collected. The flow-through was combined with excess 2H/15N-labeled GM64–237 and concentrated, and the complex was purified using SEC [pre-equilibrated in 20 mM bis-tris (pH 6.8), 150 mM NaCl, and 0.5 mM TCEP]. Fractions containing the holoenzyme complex were concentrated to 0.1 mM for NMR studies.

To generate the GM64–93:PP1α7–300 complex for crystallization, purified PP1 was incubated with microcystin-LR (MC-LR), and GM64–93 was added to a final ratio of 1:1:5 in crystallization buffer [20 mM tris (pH 8.0), 50 mM NaCl, 0.5 mM TCEP, and 1 mM MnCl2]. The GM64–93 peptide was prepared by dissolving 2 mg of peptide in 1% NH4OH before diluting with buffer [20 mM bicine (pH 9.0) and 5 mM DTT]. Final complex concentration was ~8 mg/ml for crystallization trials using vapor diffusion (sitting drop).

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