Frequency distribution of one HMM head or two HMM heads bound between two filaments in the presence of ATP. (a–c) Distribution of one (1) and two (2) HMM heads in F-actin–HMM complex in the presence of 0.2 μM ATP (319 binding events), 2.0 μM ATP (488 binding events), or 10 μM ATP (1,072 binding events). (d–f) Distribution of one (1) and two (2) HMM heads in cTFs–HMM complex in the presence of 0.5 μM ATP and low Ca²⁺ (702 binding events), 0.5 μM ATP and high Ca²⁺ concentration (545 binding events) and 2.0 μM ATP, high Ca²⁺ concentrations (440 binding events). The 0 indicates the temporarily no bindings of HMM heads to the binding sites along the length of the filaments in analyzed datasets. HMM heads are indicated by white arrows (one head bound) and red arrows (two heads bound). Scale bars, 30 nm; z-scales indicated for each HS-AFM image. Fa, F-actin.