CRYO-DIFFRACTION OF INSECT FLIGHT MUSCLE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的程序结合和比较了X射线衍射(甘油天然纤维)和薄层电子显微镜断层扫描(快速冷冻和冷冻替代纤维)来表征结晶规律性极佳的水虫飞行肌肉(IFM)中肌球蛋白交叉桥(肌肉的运动分子)的结构、排列和动态编排。揭示的X射线结果将通过在相同状态或动作下快速冻结的纤维的平行EM进行检查，以直接成像结构变化。(IFM提供了最清晰的肌球蛋白横跨任何肌肉的EM图像。)IFM的X射线图样与其他横纹肌(青蛙骨骼)的X射线图样有重要的不同，这是由于肌丝格子堆积、粗丝螺旋几何形状和全重叠肌节方案限制为~3%的长度变化所致。可以归因于肌球蛋白细丝、肌动蛋白细丝以及两者共同作用的X射线反射(~5 nm)和青蛙肌肉一样被研究得很好，但在动态(收缩)状态下的研究还不是很好。由于APS的光束质量、强度和探测器系统，对青蛙肌肉的大部分追赶研究可以在2-4年内完成。目前的建议是发展在拉伸或钙激活的收缩状态下的冷冻-捕获动态转变的冷冻衍射，以表征肌球蛋白的交叉桥作用，这些跨桥作用是力产生、受力、主动缩短和制动对强迫延长的抵抗的基础。 GUP建议的目的是评估可以提高样品寿命的冻结条件，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，即使是最弱的反射。由于肌肉系统的衍射很弱，因此必须在同步加速器上进行这项研究。据我们所知，以前还没有在肌肉上成功实现过这一点，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.