The kinetics of double-headed myosin motors bound to non-regulated actin filaments. (a) Diagram illustrating the approach used to study cooperative behavior of HMM molecules bound between two parallel filaments. (b) Structural model of actin–myosin complex for the experimental design used in the study; actin–myosin complex in rigor conditions (PDB accession no. 1M8Q) with upper and lower heads bound between two parallel actin filaments and attached by their S2 fragments (PDB accession no. 2FXO). (c) Kinetics model of actin–myosin interaction and approach used to calculate the backward and forward myosin displacements; the green dots indicate the center of mass of the head. K_T and K_-AD are the constants of the ATP binding and ADP release; K_w and K_ws are the constants of the weak biding and weak-to-strong transition, respectively. (d and e) Representative time course of binomial binding (d) and head displacements (e) calculated for the individual HMM molecules (M1–M4) at the given time for upper and lower heads of each HMM molecule in the presence of 2 μM ATP. (f) The backward and forward myosin displacements in the F-actin–HMM complex (6,589 events, ∼43 HMM molecules); data sets were fitted by sum of two Gaussians (r² = 0.99). The two peaks for backward displacement: −1.6 ± 0.2 and −4.1 ± 0.5 nm; the two peaks for forward displacement: 1.6 ± 0.2 and 4.1 ± 0.6 nm. (g) Simulated HS-AFM image of the structural model shown in b performed in Bio-AFM viewer software (Amyot and Flechsig, 2020). (h) Representative HS-AFM snapshots of HMM molecules bound between two actin filaments at the indicated times (M1–M4 shown in color code duplicated in d and e and across all of the figures), scale bar, 30 nm. Scan area: 150 × 75 nm², 80 × 40 pixels². Related to Videos 1, 2, and 3.