Fig. 5.

Illustration of initial structural evolution gating ESPT in the Ca²⁺-free GEM-GECO1. Geometric constraints from the Ca²⁺-free GCaMP2 crystal structure 3EKJ are used. An optimized ESPT pathway is delineated with an intervening H₂O molecule added (Fig. S4B) due to limited resolution of the crystal structure. To obtain an SYG chromophore, the methyl group of T223 is replaced by an H atom without further structural modification. The 170 cm⁻¹ mode facilitates ESPT with three positions depicted (DFT-calculated mode displacements are ±2° in equilibrated S₀). The bridge C—C single bond shifts from the inherent structure (gray, 125°, the bridge C = C–C angle) back (orange, 132°) and forth (cyan, 118°) to modulate H-bonding geometry in S₁ with the adjacent H₂O molecule. C, O, N, and H atoms are shown in gray, red, blue, and white, respectively. The surrounding protein environment is shown in green. Rendered using visual molecular dynamics (VMD) (49).