Fig. 5: Conformational dynamics of ATL1_cyto-TK monomers in the absence or presence of GDP revealed by intramolecular smFRET assays.

a Strategy of the intramolecular smFRET assays for ATL1_cyto-TK monomers in the absence or presence of GDP. The interaction between the His tag and biotinylated anti-His antibody was used to immobilize the protein in a streptavidin-coated microfluidic chamber. b Representative fluorescence and smFRET trajectories of intramolecular smFRET assays for ATL1_cyto-TK monomers in the absence (bottom) or presence of GDP (top). LD555 (the donor) is shown in green, LD655 (the acceptor) in red, and FRET traces are displayed as a dark blue line for FRET efficiency and pink line for hidden Markov model initialization. c, Intramolecular smFRET distributions of ATL1_cyto-TK monomers in the presence (left) or absence of GDP (right). All individual FRET values with the number of molecules (Nm) displayed were compiled into a conformation-population FRET histogram (gray lines) and fitted into a four-state GaussAmp distribution as ultra-low-FRET state (centered at ~0.18), medium-FRET state (centered at ~0.41), high-FRET state (centered at ~0.63), and ultra-high-FRET state (centered at ~0.83) shown in red, blue, purple, and green lines, respectively. Each bar height represents the normalized count (%). The length of the error bar represents the normalized SD of a Poissom distribution from the count. d TDP plots under different conditions. Average FRET values before transition versus after transition are displayed as a 2D chart (scale at right) with the number of transitions (Nt) shown above. e Kinetic analysis of the ATL1_cyto-TK monomers in the absence or presence of GDP. The differences in transition rate constants shown in Supplementary Fig. 15 between GDP-bound and nucleotide-free ATL1_cyto-TK are shown. f Relative free energy model of the conformational dynamics of ATL1_cyto-TK in the absence (bottom) or presence of GDP (top). The ∆G of the ultra-low-FRET state compared with the medium-FRET state, high-FRET state, and ultra-high-FRET state is roughly scaled and marked below the corresponding states. Source data are provided as a Source Data file.