Protein biochemistry. Four different CLZ containing constructs of varying lengths were generated for both CNGA1 and CNGA3 ( Supplementary Fig. 3) . Each of these eight constructs was subcloned into two bacterial expression vectors, pNGFP_BC and pCGFP_BC (kindly provided by Dr. E. Gouaux), creating aminoterminal and carboxy-terminal GFP fusion proteins respectively. Each of these 16 constructs was screened by FSEC as previously described 28 . Briefly, 5 ml bacterial cultures were induced, spun down, and resuspended in 800 µl of 150 mM KCl, 30 mM HEPES, pH 7.5 containing 2.5 µg ml − 1 DNAse and cOmplete protease inhibitor tablets (Roche). The cells were lysed with a probe sonicator; the lysate was cleared by centrifugation (1,700 × g for 30 min); and 100 µl of the supernatant was loaded on a Superdex 200 10/300 GL column (GE Healthcare) mounted on an FPLC system with a fluorescence detector set for detection of GFP fluorescence. Four candidates from the FSEC screen (CNGA1#621-690, CNGA1#621-667, CNGA3#626-694, and CNGA3#626-672) were subjected to further analysis. For large scale protein preparations, the CNGA1 and CNGA3 channel fragments were subcloned into the pETGQ and pETM11 vectors, respectively, creating amino-terminal poly-His fusion proteins. For the CNGA3#626-672 construct, the NcoI site at the amino terminus was deleted using PCR-based methods. Two litre bacterial cultures were grown to mid-log phase and induced with 1 mM IPTG overnight at 18 °C. The cultures were spun down and resuspended in 150 mM KCl, 30 mM HEPES, pH 7.5 containing 1 mM AEBSF, 2.5 µg ml − 1 DNAse and cOmplete protease inhibitor tablets (Roche). Cells were lysed with an EmulsiFlex C-5 homogenizer (Avestin), and the lysate was cleared by centrifugation at 131,000 × g for 45 min at 4 °C. The proteins were then purified on a Ni 2 + affinity resin column (HisTrap HP, GE Healthcare). The octahistidine tag was removed by thrombin (1) (1) (2) (2) or TEV cleavage, and the proteins were further purified on an anion or cation exchange column (HiTrap Q HP or HiTrap SP FF, GE Healthcare) and concentrated.