Fig. 2

Characterization and structure of K-GECO1. a Fluorescence excitation and emission profile of K-GECO1 in the presence and absence of Ca²⁺. b Ca²⁺ titration curve of K-GECO1. c K-GECO1 effective two-photon fluorescence excitation spectra in Ca²⁺-saturated (red symbols) and Ca²⁺-free (blue symbols) states. Ratio of the K-GECO1 two-photon excitation fluorescence Ca²⁺-saturated/Ca²⁺-free signals as a function of wavelength (black symbols, plotted on right y-axis). d Two-photon molecular brightness of K-GECO1, R-GECO1, and mCherry with excitation at 1060 nm using various laser powers. Overall protein structures of genetically encoded Ca²⁺ indicators: e K-GECO1 (PDB: 5UKG), f R-GECO1 (PDB: 4I2Y [19]), g RCaMP (PDB: 3U0K [19]), and h GCaMP6 (PDB: 3WLD [60]), with ckkap colored in magenta, RS20 in yellow, the CaM N-lobe in dark blue, and the CaM C-lobe in cyan. Zoom-in view of the interactions between key residues and the chromophore: i K-GECO1, j R-GECO1, k RCaMP, and l GCaMP6. Supporting numeric data are provided in Additional file 8. PDB Protein Data Bank