We produced numerous additional GCaMP variants and tested them in automated neuronal assays (Fig. 1). With the aim of improving sensitivity, we focused mutagenesis on the interface between cpGFP and CaM at 16 amino acid positions, some mutagenized to near completion (Fig. 1a, Supplementary Table 5)16. Mutations were made at 18 additional sites, notably at the M13/CaM interface which can affect calcium affinity28 (A317) and in CaM (R392)16 (Fig. 1a).
