This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our program correlates X-ray diffraction (glycerinated native fibers) and thin-section EM tomography (fibers quick-frozen &freeze-substituted) to characterize structure, arrangement and dynamic choreography of myosin crossbridges (motor molecules of muscle) in a waterbug insect flight muscle (IFM) of unexcelled crystalline regularity. X-ray results will be supported and checked by parallel EM on fibers quick-frozen during the same state or maneuver, to image structures directly. (IFM gives the clearest EM images of myosin crossbridges of any muscle.) IFM X-ray patterns differ significantly from those of other striated muscles (frog skeletal) studied by XRD, reflecting different filament lattice packing, filament helical geometry, and full-overlap sarcomeres limited to 3-5% length changes. X-ray reflections (to ~5 nm) attributable to myosin filaments, actin filaments, and both jointly, are as well worked out as in frog muscle, and we are catching up regarding dynamic (contraction) states. Thanks to the beam quality, intensity and detector systems at APS/BioCAT, much of the catch-up with frog muscle studies can be done in another 2 years. We propose here to measure diffraction patterns from wet & frozen IFM, relating graded x-ray signals of crossbridge binding and tropomyosin movement to [Ca2+]-dependent force levels &to time-dependent rise of stretch-activated force, dissect incremental crossbridge braking and force-holding structural responses, time-resolve crossbridge angle changes and myofilament elasticity by x-ray interference (collaborator HE Huxley), and to correlate the findings of SAXS cryo-diffraction and EM of the same or parallel quick-frozen IFM fibers.
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