Tissue‐Specific Extraction and Structural Elucidation of Actin‐Tropomyosin‐Myosin Complexes from Human and Rodent

ABSTRACT Muscle contraction and cellular motility depend on the complex interplay between myosin, actin, and associated proteins. Disruptions in these interactions are linked to various human diseases, including muscular dystrophies and cardiac conditions. In this study, we developed a tissue‐extraction protocol to purify the actin–tropomyosin–myosin (ATM) complex and filamentous actin (F‐actin) directly from human and mouse left ventricles, as well as from rat skeletal muscles. Utilizing cryo‐electron microscopy (cryo‐EM), we resolved the structures of the ATM complexes and F‐actin derived from these tissues. Additionally, we extracted ATM complexes from mice carrying the hypertrophic cardiomyopathy (HCM) mutation R404Q and demonstrated how this mutation alters the formation of ATM complexes and the structural configuration in myosin. Our approach offers a general method for isolating intact ATM complexes directly from various mammalian tissues, providing insights into the structural basis of ATM complex formation and regulation in muscle function and disease.