Fig. 4. Dual binding modes are observed by smFRET.

a The C-terminus of Coh (position 154) was labeled with the FRET donor Cy3b (shown in magenta) while the C terminus of the XMod-Doc (position 199) was labeled with the FRET acceptor AF647 (shown in orange). The donor–acceptor distance was smaller in binding mode A (3.5 nm) than the binding mode B (4.9 nm), giving rise to a higher FRET efficiency when the complex formed in binding mode A. b FRET efficiency histograms measured using Cy3b-labeled WT XMod-Doc (top), BM^A-KO (middle), or BM^B-KO (bottom) complexed with AF647-labeled Coh. Efficiency histograms were fitted with single or double Gaussian distributions.