Extended Data Fig. 9: Ligand binding in wFAP1.1 and tmFAP1.1 structures.

a, HBC599 docked to the crystal structure of wFAP1.1 (white ribbon) by Autodock Vina. The top docking output is shown in green, highly consistent to that in the crystal structure (white). b, Potential binding mode for HBC620 to wFAP1.1. HBC620 was docked to the protein part of the wFAP1.1 crystal structure (white). The binding mode of HBC620 in the top output (green) closely resembled that of HBC599 (white). (c-d), Mutations introduced in the binding pockets of wFAP1.1 (c) and tmFAP1.1 (d) abolished or reduced the fluorescence activation, in agreement with the structures of wFAP1.1 and tmFAP1.1. E. coli cells expressing the mutants were analyzed by cytometry using the same method as directed evolution experiments. All samples were incubated with 200 nM HBC599 for the same amount of time. N = 3000 different cells of each mutant were used for analysis. The center dot denotes the median value. The upper and lower bounds of box denote the 25th and 75th of the data. Whiskers extended the range to the 10th to 90th percentile. (e-f), Correlation between pLDDT in structure prediction models and B-factor in experimental structures. Yellow dots denote pocket residues, while blue dots denote the remaining residues. The Pearson correlation coefficient (r) and p-value (p) are presented for all residues and pocket residues, respectively (Methods). Data plotted for e, wFAP1.1. f, transmembrane region of tmFAP-BRIL.