Comparison of cryo-EM-based atomic models of myosin-free and myosin-decorated actin-tropomyosin filaments shows a clear azimuthal shift in tropomyosin position between corresponding C- and M-state thin filaments. Arg 369 and Glu 370 on the tip of the Loop-4 of the myosin-head likely drive C-state tropomyosin to its M-state position, while Loop-4 along with the cardiomyopathy (CM) motif and other surface loops on the myosin-head elicit the tight binding characterizing the M-state myosin-head interaction with actin. These cryo-EM-based models of myosin-decorated thin filaments were generated from filaments saturated with myosin-S1, i.e., here all actin subunits along thin filaments contain a bound myosin-head. However, this arrangement is non-physiological, since in actively contracting muscle, few myosin-heads attach to neighboring actin subunits along thin filaments at the same time. Thus, the collective effect of multiple interacting myosin-heads on the positioning of tropomyosin may be amplified artificially over what is physiologic. In the current cryo-EM study, we controlled the level of S1-binding to actin-tropomyosin. We selected and analyzed filament segments where only a single myosin-head bound to stretches of myosin-free actin-tropomyosin. The single myosin-heads and associated actin-tropomyosin were successfully aligned to each other yielding a 5 Å resolution 3D-reconstruction. Tropomyosin on the actin helical strand containing the bound myosin-head occupied the M-state position, which was found to be indistinguishable from the position of tropomyosin in fully decorated filaments. In contrast, tropomyosin, on the opposite side of double-helical actin that is “untouched” by myosin, occupied the C-state position. Our new precision will allow us to determine structurally the cooperative unit size for the myosin-induced C- to M-state tropomyosin transition and to investigate factors that may lead to cross-strand tropomyosin communication.
Karpicheva et al. (Sun,) studied this question.