CRYO-DIFFRACTION OF INSECT FLIGHT MUSCLE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 我们的程序结合和比较X射线衍射（甘油化天然纤维）和薄切片EM断层扫描（纤维速冻和冷冻取代）来表征肌球蛋白横桥（肌肉的马达分子）的结构，排列和动态编排在一个水蝽昆虫飞行肌肉（IFM）的无与伦比的结晶规律。 揭示X射线结果将通过在相同状态或动作期间快速冷冻的纤维的平行EM来检查，以直接成像结构变化。 (IFM给出了任何肌肉的肌球蛋白横桥的最清晰的EM图像。 来自IFM的X射线图案与通过XRD研究的其他横纹肌（青蛙骨骼肌）的X射线图案在重要方面不同，这是由于细丝晶格堆积、粗细丝螺旋几何形状和限制于~3%长度变化的全重叠肌节方案的差异。 肌球蛋白丝、肌动蛋白丝以及两者共同产生的X射线反射（~5 nm）与青蛙肌肉中的一样，但在动态（收缩）状态下还没有得到很好的研究。 由于APS的光束质量、强度和探测器系统，大部分青蛙肌肉研究可以在2-4年内完成。 目前的建议是开发冷冻衍射的冻结捕获的动态转换在收缩状态激活的拉伸或钙的肌球蛋白过桥行动的特点，根据力的产生，受力，主动缩短和制动阻力被迫延长。 本GUP建议的目的是评估可以提高样品寿命的冷冻条件，以便在辐射损伤开始之前从小样品中获得最高分辨率的衍射图案，显示甚至最弱的反射。由于肌肉系统衍射的弱点，这项研究必须在同步辐射源上进行。据我们所知，这在肌肉中还没有成功实现，并且可能会导致新的发现。

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 combines & compares 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. Revealing X-ray results will be checked by parallel EM of fibers quick-frozen during the same state or maneuver, to directly image the structural changes. (IFM gives the clearest EM images of myosin crossbridges of any muscle.) X-ray patterns from IFM differ in important ways from those of other striated muscles (frog skeletal) studied by XRD, owing to differences in filament lattice packing, in thick filament helical geometry, and a full-overlap sarcomere scheme limited to ~3% 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, but not yet so well studied in dynamic (contraction) states. Thanks to the beam quality, intensity and detector systems at the APS, much of the catch-up with frog muscle studies can be done in 2-4 years. The present proposal is to develop cryodiffraction of freeze-trapped dynamic transitions during contractile states activated by stretch or calcium to characterize myosin crossbridge actions that underlie force generation, force bearing, active shortening and braking resistance to forced lengthening. The purpose of this GUP proposal is to evaluate freezing conditions that can enhance sample lifetime so that diffraction patterns of highest resolution, showing even weakest reflections, can be obtained from small specimens before radiation damage sets in. Because of the weakness of the diffraction from the muscle system this study must be done on a synchrotrron source. As far as we know this has not been successfully achieved in muscle before and may be expected to lead to novel findings.