Myofibril structure of insect flight muscle (IFM) is being studied by coordinated use of electron microscopy, X-ray diffraction, quantitative microscopy and biochemical analysis, with emphasis on the number, arrangement and behavior of myosin crossbridges. Crossbridges are the primary movers in muscle contraction and most non-muscle motility, and their highly ordered lattice arrangement in IFM permits averaging methods to extract crossbridge structure in different states which can be related to different phases of their ratchet-like cycle. 3-D computer reconstructions from tilt views of thin sections are proceeding, aided by optical diffraction and image filtering, to extract maximum information from muscle processed by specially optimized fixation-embedding techniques. Still better preservation will be sought by X-ray monitoring of new procedures. Thin filament and thick filament structure are to be explored separately in I- and H-bands of stretched sarcomeres, an obvious preparation but one not properly developed until recently. The 3-D work is collaborative, as are co-ordinated studies of crossbridge orientation by EM/X-ray and spin-labelling, and antibody localization of troponin within the crossbridge lattice. All these approaches will be applied to several crossbridge states, certainly to rigor, ATP-relaxed, AMPPNP-induced and S-1 loaded, and possibly to the Ca-ATP state and to thiol-modified and to carbodiimide cross-linked fibers. In a different approach, quantitative microscopy (EM and interference), buoyant density banding in Percoll, and X-ray diffraction measurement of lattice spacing will be all be co-ordinated in studies of dry mass content of myofilaments and fibrils, to produce mass distribution profiles of insect and rabbit sarcomeres, ascertain absolute protein concn in sarcomere cross-bands, determine actin occupancy by crossbridges in various states, and to try to resolve an anomalous discrepancy (found also in vertebrate muscle) between filament mass and whole fibril mass.
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