Supplementary Figure 1: The topology of mIFP (i.e., bacteriophytochrome).

Scheme is based on alignment of the sequence of mIFP (320 residues; GenBank accession number AKH03689.1; Nat. Methods 12, 763–765, 2015), with the crystal structure of the chromophore-binding domain of Deinococcus radiodurans BphP (PDB 2O9B; J. Biol. Chem. 282, 12298–12309, 2007). Representation is adapted from Takala et al. (Nature 509, 245–248; 2014), with β-strands represented as arrows and α-helical regions represented as cylinders. The PAS domain is colored in light green, and the BV-binding GAF domain is colored in light blue, as in Fig. 1a,b. The approximate position of the bound BV is represented by a magenta structure. Numbers at the ends of β-strands correspond to mIFP numbering (see Supplementary Figs. 2c, 3 and 4), based on alignment with the crystal structure. To engineer NIR-GECO1, 5 residues (171–175, DEEGN) in the loop between the first two β-strands of the GAF domain were initially replaced with a 182-residue CaM-RS20 domain (a 3-residue linker followed by 147-residue CaM followed by a 5-residue linker followed by 23-residue RS20 followed by a 4-residue linker). Systematic optimization of the insertion site to improve the Ca²⁺-dependent fluorescence change led to the deletion of residues 176G and 177E of mIFP, resulting in an overall replacement of 7 residues (171–177, DEEGNGE) with the CaM-RS20 domain.