Extended Data Fig. 10: Mapping of mutations introduced into b11 to yield the final brighter variants, biophysical characterization of mFAP1 and 2, and epifluorescent images.

a, Sequence alignment of b11-based DFHBI-binding fluorescence-activating proteins. Orange boxes indicate mutations or loop insertions introduced by computational design; purple boxes highlight mutations rationally introduced on the basis of the deep mutational scanning maps (Extended Data Figs. 7, 8); green boxes indicate mutations or loop insertions that were incorporated during combinatorial library selections; K40V and K54Y in light blue boxes were introduced to help crystal formation (Extended Data Fig. 9h, i). Despite having hydrophobic residues on the surface, mFAP2 remains soluble at 150 mg ml⁻¹. b, Mutations in the mFAPs mapped on the design models. Common mutations in all three mFAPs are highlighted in bold. c, Absorbance spectra for DFHBI, and the mFAP1–DFHBI and mFAP2–DFHBI complexes (n = 4 biological replicates with similar observations). d, Extinction coefficient determination for DFHBI at 418 nm. e, Normalized absorbance and fluorescence spectra of the mFAP1–DFHBI and mFAP2–DFHBI complex. Data are representative of two biological replicates with similar observations. f, g, Widefield epifluorescence (bottom) and brightfield (top) images of E. coli and yeast cells with 20 μM DFHBI. Untransformed E. coli Lemo21 cells (f, left, n = 2 biological replicates with similar observation) and yeast EBY100 cells displaying ZZ domain (g, left, n = 2 biological replicates with similar observation) were treated with the same amount of DFHBI and imaged in the same way (1000 mA 470-nm LED and 200-ms exposure time).