(A) Representative micrographs of actin filaments decorated with nucleotide free myosin VI (left), Mg-ADP myosin VI (middle), and in the absence of myosin (right). Scale bar, 100 nm. (B) Fourier Shell Correlation (FSC) curves for reconstructions of actin-MyoVI rigor (left) and ADP (right) with RNA (grey), without RNA (black), and combining the two datasets (magenta). (C) Cartoon ribbon diagram of the engineered myosin VI used in this study. (D) FSC curves for reconstructions of actin-myoVI rigor state (left), ADP state (middle), and bare actin (right). FSC curves approximately corresponding to different domains were generated by radially masking reconstructions at 120 Å (full reconstruction, magenta), 90 Å (truncated MD to exclude lever arm and converter, dark magenta), and 40 Å (actin, light blue). (E) Reconstructions for actin-myoVI rigor (left), actin-myoVI ADP (middle), and bare actin (right) colored according local resolution as determined by ResMap. Radii corresponding to S1D indicated. (F) View of the HR rigor MDFF model (magenta) and the rigor-like state initial model (2BKI, grey) superimposed in the reference frame of the actin filament (light gray density). The superposition was generated based on the Cα coordinates of the full motor domain. Rigor U50, magenta; rigor L50, dark magenta; 2BKI U50, dark grey; 2BKI L50, light grey; actin density, white. Arrows denote direction of domain centroids (spheres) from 2BKI to HR rigor MDFF. Centroids were determined for the U50 (residues 180–206, 229–397, and 405–441) and L50 (residues 467–597 and 638–661) domains.