Extended Data Fig. 2: Results of cultured neuron 1-AP field stimulation screen (n = 813 constructs, 647 with detectable 1-AP responses; Methods).

All results are normalized to in-plate GCaMP6s controls (blue line) and listed in ranked order (increasing for peak ΔF/F₀, decreasing for all others) from each screening round. Other relevant control constructs (n = 9) were screened side-by-side (right panels). Sensor engineering took place over seven rounds: Round 0 (r0): Graft peptides (n = 29 constructs). Round 1 (r1): Screen linkers (n = 64 constructs). Round 2 (r2): Site-saturation mutagenesis of 16 interface positions: 7 in ENOSP, 4 on cpGFP, and 5 on CaM (n = 304 constructs). Round 3 (r3): Combination of beneficial mutations to date (n = 69 constructs). Round 4 (r4): Site-saturation mutagenesis of 10 additional CaM positions surrounding ENOSP and of 3 residues on linker1 (n = 272 constructs). Graft mutations from FGCaMP. Round 5 (r5) and 6 (r6): Two additional rounds of combination of beneficial mutations (n = 25, 51 constructs respectively).