Exciting recent progress promises continued revelations from time-resolved structural analysis of motor molecules in muscle, to clarify individual motor actions as well as their integrated ensemble performance in whole muscles. High paracrystalline order of glycerinated insect flight muscle (IFM) from giant waterbug Lethocerus gives distinctive X-ray diffraction (X-ray) from each physiological state, and allows unexcelled thin-section electron microscope (EM) images of myosin crossbridges. Comparing image Fouriers with native X-ray patterns measures and validates fidelity of thin-section EM. New synchrotron X-ray beamlines and detectors allow new methods. We will coordinate time-sliced synchrotron X-ray diffraction with EM 3-D reconstructions (3-Ds) from quick-frozen fibers to integrate mechanical with structural data for millisecond-timed snapshots of the myosin power stroke. 3-D analysis of crossbridge diversity in active states is based on 3-D EM tomography, which can present non-averages structures, but also allows partial or full averaging, as well as 3D correspondence analysis to classify and count different crossbridge shapes. Load-induced distortions of IFM high-force crossbridges in active, rigor and AMPPNP states will be mechanically synchronized by step/ramp length changes and studied by X-ray, 3D EM of cryo- and chemfixed fibers, and fitting of crystal-structure models of myosin and actin. Force generation schemes will be modeled from these fittings. Low-force (pre-powerstroke) active bridges, to be favored at high (Ca2+) by cooling, high (Pi), and Pi analogs (BDM, vanadate, AlF4-, BeFx), will be compared with high-force bridges using X-ray, 3D-EM and fitting of atomic models. X-ray cryodiffraction of quick-frozen IFM fibers will be developed to protect against high-flux radiation damage while recording high resolution exposures, and to freeze-capture stable, evolving and maybe dynamic states. Myosin's S1-S2 junction at the crossbridge origin in IFM will be probed by low-salt lattice swelling and high-salt loosening of the thick filament backbone. Thick filament and A-band structure in relaxed IFM will be collaboratively modeled from our X-ray data at 2o, 20o and 40o (normal flight temperature in vivo), using our supporting EM studies to generate model geometry.
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