Analysis of conformational dynamics of single α_Lβ₂. (A–D) Switching times (A and B) or times to switch (C and D) of α_Lβ₂ bending or unbending in Ca²⁺Mg²⁺ or Mn²⁺ were analyzed by distributions in semilog plots (A and C), from which the expected (closed bars) and mean (open bars) values were calculated (B and D). Numbers of measurements and p-values of the Student’s t test are indicated. Data in A and C are linearly fitted by solid lines with the corresponding colors. (E–H) Semilog plots of survival frequencies of unbending (E and G) and bending (F and H) events versus switching time t_sw (E and F) or time to switch t₀ (G and H). A straight line was fit to data for each force bin (indicated) to estimate the expected t_sw or t₀ from the negative reciprocal slope. (I and J) Force-dependent switching time (I) and time to switch (J) of α_Lβ₂ in Mn²⁺ (left ordinate). Data and their linear fits (solid lines) to the expected (Δ and ∇ calculated from E–H) and mean (○ and □) values for bending (cyne) and unbending (red) are shown. The levels of significance of the differences between the slopes and 0 are indicated by the p-values. The ratio of time to unbending to time to bending, plotted as a pink solid curve (right ordinate), shows the force-dependent equilibrium constant of the bent state. (K) Force-dependent frequencies of bending or unbending of α_Lβ₂ in the position-clamp phase measured in Mn²⁺. Error bars in B, D, I, and J represent 95% confident range of the slope (for expected values) or SEM (for mean values).