Probability of myosin heads binding to the regulated cTFs. (a and c) Fitting the simulated HS-AFM images (Amyot and Flechsig, 2020) of the cTFs at Ca²⁺- free (a) and Ca²⁺-bound (c) states into the cTFs molecular structures, scale bar, 2.5 nm. PDB accession nos. 6KN7 and 6KN8 for Ca²⁺ free and high Ca²⁺ states, respectively. (b and d) Representative HS-AFM snapshots of HMM molecules bound between two cTFs in the presence of 0.5 μM ATP and either low Ca²⁺ (b, scan area: 200 × 200 nm², 120 × 120 pixels²) or high Ca²⁺ (d, scan area: 200 × 200 nm², 120 × 120 pixels²) concentrations at the indicated times. Scale bars, 30 nm. Note that the number of bound heads increase with time in d. (e–g) Probabilities of the HMM heads binding to the regulated cTFs in the presence of 0.5 μM ATP and low Ca²⁺ (492 events) or 0.5 μM ATP and high Ca²⁺ concentrations (2,830 events; f) or 2 μM ATP and high Ca²⁺ concentration (1,839 events; g) with the corresponding average fractional occupancy versus time shown in the right panels. (h) Frequency distributions for the number of myosin heads attached to four binding sites along all studied thin filaments over time in the activating conditions (n = 7 experiments, ∼35 HMM molecules, 932 events). Mean number (±95% CI) of attached myosin heads relative to the four available binding sites given in the inset text. Full lines and dashed lines in the right panels in e–h represent regression lines and 95% confidence intervals. The analysis suggested a decline in the number of myosin molecules with time, but to a lower degree compared to the F-actin–HMM complex. (i) Cooperative probability of binding of myosin heads in F-actin–HMM and cTFs–HMM complexes (n = 7: cTFs–HMM, n = 8: F-actin–HMM, ∼40–50 HMM molecules in each data sets were analyzed; the data points shown as mean value ± 95% CI). Related to Video 4.