Extended Data Fig. 1: The engineering process leading to the GRABACh3.0 sensor.
From: An optimized acetylcholine sensor for monitoring in vivo cholinergic activity
a, Schematic illustration depicting the predicted structure of the generic GRABACh sensor, with the linker region between the receptor (M3R) and cpEGFP magnified at the right and shown in magenta. The crystal structures are from protein database (PDB) archive (PDB ID: 4DAJ for M3R; PDB ID: 3EK4 for cpGFP). b, Site-directed mutagenesis of residues in the N and C termini of the linker region. The numbers indicate amino acid positions in the linker region (the first on N-terminus as N1, and the first on C-terminus as C1). The candidate with the best response is shown in a black circle and is called ACh2.5, with the C4 residue mutated to K; this candidate is used for further engineering steps. c, Left: crystal structure of the cpEGFP moiety in the ACh3.0 sensor; targeted residues for mutagenesis screening are indicated in green and the corresponding amino acid labeled on the structure. Right, the fluorescence response of the indicated mutant candidate sensors is shown on top, with the sequences of the best-performing candidates on the bottom; the relative size of each letter reflects the probability of that amino acid in the sequence. The residues are named by the amino acid followed by the position in cpGFP (the first amino acid in cpGFP as N1). The crystal structures are from protein database (PDB) archive (PDB ID: 3EK4 for cpGFP). d, The fluorescence response of each candidate ACh sensor with combined mutations from the best-performing sites in the linker and cpEGFP. Each point is calculated from the average of >100 cells. e, left, illustration of the ligand binding pocket in M3R, which was mutated from W to A. Right, fluorescence image of HEK293T cells expressing ACh3.0-mut. f, The fluorescence response of ACh3.0 and ACh3.0-mut to indicated concentration of ACh applied (n = 3 wells for each point, with each well averaging >100 cells). Scale bar represents 10 μm. All data are shown as mean value ± SEM, with the error bars or shaded regions indicating SEM.
