The S. cerevisiae calmodulin gene CMD1 was cloned from genomic DNA into a bacterial expression plasmid with a sequence encoding His6-TEV situated at the 5′ end to generate pDD2743. pDD2743 was transformed into Rosetta E. coli, optimized for expression (Novagen). A saturated overnight culture in LB (10 g/L Bacto tryptone, 5 g/L Bacto yeast extract, 10 g/L NaCl) was used to inoculate a 1-L culture in LB to OD600 = 0.1. Cells were grown to OD600 = 0.6–1, induced with 0.5 mM IPTG for 5 h at 37°C, pelleted at 4,225 × g for 20 min at 4°C in a Sorvall SLA-3000 (fixed angle) rotor, washed with cold 20 mM HEPES pH 7.5, and re-pelleted at 2,250 × g for 10 min at 4°C in a Jouan CR3i (swinging bucket) centrifuge. Cell pellets were flash-frozen in 45 ml lysis buffer (20 mM HEPES pH 7.5, 1 M KCl, 20 mM Imidazole). Upon thawing, cells were lysed by sonication, 2 mg DNase I (Roche) and Triton X-100 to 1% were added, and the resulting lysate was incubated on ice for 30 min, then spun at 92,000× g for 25 min in a Beckman Type 70 Ti rotor. The supernatant was loaded onto a 1-ml HisTrap HP column (GE healthcare) pre-equilibrated with binding buffer (20 mM HEPES pH 7.5, 500 mM KCl, 20 mM imidazole). The column was washed with 20 ml binding buffer, and Cmd1 was eluted using a 30 ml linear gradient from 0–100% elution buffer (20 mM HEPES pH 7.5, 500 mM KCl, 500 mM imidazole). Fractions containing Cmd1 were pooled, Cmd1 was cleaved from His6 with TEV protease, and dialyzed overnight at 4°C into a low salt buffer (10 mM Tris pH 7, 25 mM NaCl, 2 mM MgCl2, 5 mM DTT). Following dialysis, purified, cleaved Cmd1 was bound to a MonoQ column and eluted using a 10 ml linear gradient from 0–70% high salt buffer (10 mM Tris pH 7, 1 M NaCl, 2 mM MgCl2, 5 mM DTT). Fractions containing Cmd1 were pooled, dialyzed into KMg50 buffer (60 mM MOPS pH 7, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 5% glycerol), and stored at −80°C.