How muscle contracts has been a long-standing question. Despite major advances in this area, how
muscle relaxes is still not fully understood. Contraction occurs by the sliding of myosin-containing thick past
actin-containing thin filaments, powered by myosin heads, motors that produce sliding force, fueled by ATP.
Relaxation occurs when thin filaments are switched off so heads cannot bind to produce force, leaving the idling
heads to organize themselves helically in the thick filament. What is currently known about the role of thick
filaments in relaxation? On the structural side, low-resolution models of cardiac (mouse, human) and skeletal
(tarantula) thick filaments have been achieved, but their atomic structure remains unsolved. On the energetics
side, the energy consumption of relaxed skeletal muscle revealed a surprising phenomenon, so-called super-
relaxation (SRX) that greatly reduces ATP consumption. A widely accepted view associates this ubiquitous and
fundamental energy-saving state with the unique way myosin’s two heads fold together in the relaxed tarantula
filament—the so-called interacting-heads motif (IHM), found across the animal kingdom, which structurally
inhibits both heads, switching off their activity. Regardless of its appeal, this SRX=IHM hypothesis has not been
proved, and recent ATP turnover results suggest, instead, association of SRX with a specific myosin head
conformation. Elucidating this puzzle is crucial to understanding how muscle relaxes, how it malfunctions in
disease and how therapeutic drug treatments work. The solution requires determination by cryo-EM of the atomic
structures of the thick filament and myosin molecules from muscle. Here, we propose to determine the structures
of skeletal myosin molecules and filaments, far less studied than cardiac. This will allow us to dissect how key
IHM interactions constrain activity of the two heads, shutting them off, thus conserving ATP in relaxation. We will
use single particle EM and cryo-EM to define the structural basis of the SRX state at near-atomic level in thick
filaments and myosin molecules from rabbit skeletal muscle. By comparing with tarantula, which shows tenfold-
greater energy-saving (hyper-relaxation, HRX), we will gain deeper insight into the mechanism of ATPase
inhibition. And we will use EM and X-ray diffraction to investigate how therapeutic drugs alter the IHM.
Aim 1 will define the structural basis of SRX in skeletal thick filaments by revealing their near-atomic
cryo-EM structures. Aim 2 will define the structural basis of SRX in skeletal myosin heads and heavy meromyosin
molecules by assessing: (A) if SRX results from a specific head conformation, and (B) if the IHM correlates with
the SRX state. Aim 3 will reveal the structural impact of drugs on skeletal thick filaments and myosin molecules.
Despite the vital role of SRX in skeletal muscle relaxation, its structural basis and relation to the IHM and
to other thick filament proteins (MyBP-C, titin) remains unknown. Our studies will reveal the IHM structure in
skeletal thick filaments and myosin molecules, clarify its association with the SRX state and with MyBP-C and
titin, and provide critical insights into the molecular basis of relaxation and the influence of therapeutic drugs.