Electron Tomography of Relaxed Lethocerus Flight Muscle Reveals Thin Filament Binding of Disordered “Blocked” Heads

Abstract

When relaxed flight muscle fibers from Lethocerus indicus are visualized by thin section electron microscopy, they typically show pronounced shelves of density spaced at 145 Å intervals, dubbed crowns, with occasional contacts between what appear to be myosin heads and the troponin complex preferentially, but not exclusively. Recent cryoEM studies on the structure of relaxed, isolated thick filaments from Lethocerus flight muscle indicates that the myosin heads are arranged in an interacting heads motif (IHM) with a novel orientation with respect to the filament axis. The IHM free head is positioned tangential to the backbone surface with the blocked head extending to high radius and apparently more dynamic than the free head. As such, the blocked head might be the myosin head seen contacting the thin filaments in EMs of relaxed muscle fibers. The stretch activation response in Lethocerus flight muscle occurs at a submaximal [Ca2+] concentration requiring some other structure to produce complete activation. Here we have used electron tomography and subvolume averaging to investigate the distribution of myosin head-thin filament attachments in sections of relaxed fibers to determine their thick filament origin and thin filament destination. Class averages of thick filament crowns (145 Å axial repeats) show a mixture of different conformations of the IHM. Class averages of thin filaments resolve actin subunits and troponin well. In this study, we are trying to investigate the statistics of different IHM conformations in a quantitative way as well as the in situ details of the free and blocked head conformations in relaxed and partially relaxed IHMs. Supported by NIH.