Myosin-Actin Structural Ensemble Modeling

The RosettaCM hybridization protocol (33 (link), 51 (link)) was used to build structural ensembles based on multiple structure templates (Fig. 1 B and C). First, the query myosin–actin sequence is threaded onto each individual template. Rosetta uses Monte Carlo sampling to produce hybrid-template models by recombining template segments in Cartesian space and de novo building unaligned regions in torsion space. Then, the model geometry is further improved by optimizing local structure, e.g., segment boundaries and loops. In this process, conformations away from the starting templates are able to be explored through MC sampling with local fragment superposition and energy minimization moves. The high-score models are inspected and selected for MD simulations. Fig. 1C shows the case for the PPS state. For the rigor and ADP-bound states, PDB 5H53 and PDB 6C1D were used as the templates for the myosin–actin complex, respectively. The ATP hydrolysis products (ADP+Pi) were explicitly incorporated at the active site of the PPS myosin. Tropomyosin and troponin were not included in the models. Modeling details are described in SI Appendix.