Fig. 6: Myosin (HMM) molecules simultaneously attached to two different actin filaments in the presence of ADP and either metavanadate or para-aminoblebbistatin.

a Atomic models of myosin S1-ADP (PDB 3I5F), S1-ADP-VO₄ (PDB 1QVI), and S1-ADP-Blebbistatin (PDB 6Z7U with lever arm in pre-powerstroke configuration) and hs-AFM images of myosin heads simultaneously bound to two different actin filaments in the presence of ADP, ADP-metavanadate and ADP-para-aminoblebbistatin (PAB); scale bars: 30 nm. b Lever arm angles plotted as a dynamic change of lever arm configuration collected for 10 s. Time resolution: 150 ms/frame (6.7 fps). Red: F-actin-HMM-ADP (number of events: 439). Blue: F-actin-HMM-ADP-metavanadate (number of events: 272). Green: F-actin-HMM-ADP-PAB (number of events: 266). The border between purple and blue areas indicates the mean angle for the lever arm in the ADP configuration obtained from Gaussian distributions. c Molecular models of the pre-powerstoke (PPS, presence of ADP-metavanadate or ADP-PAB) and post-powerstroke (POST, presence of ADP) states with the experimentally measured difference in lever arm configuration. d Data from three independent experiments for conditions with ADP (1–10 µM), number of events: 848 ADP (10 µM) + metavanadate (100 µM), number of events: 642 or ADP (10 µM) + PAB (10 µM), number of events: 339 pooled together, showing distinctly different distributions of lever arm angle between ADP: 51.0° ± 13.42° (SD), r² = 0.99; ADP-metavanadate: 68.6° ± 13.8° (first peak), 99.2° ± 6.7° (second peak), r² = 0.99; and ADP-PAB: 53.6° ± 8.3° (first peak), 87.7° ± 16.6° (second peak), r² = 0.98. Time resolution: 150 ms/frame (6.7 fps). The averaged difference in the lever arm position between the strongly bound (post-powerstroke) and weakly bound (pre-powerstroke) states for ADP-metavanadate was: 99.2 ± 6.7°–68.6 ± 13.8° = 30.6 ± 15.3° and for ADP-para-aminoblebbistatin: 87.7 ± 16.6° – 53.6 ± 8.3° = 34.1 ± 18.6°, respectively. Data from Supplementary Movies 2, 4, 5, 7, 11, 15, 17.