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。所有提出的X射线实验，无论何时揭示，都将与在相同状态或动作中快速冻结的纤维的平行电磁实验相匹配，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。目前的建议是继续我们最近关于横桥制动动作的新工作。 这一快速访问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更高分辨率的衍射图。由于来自肌肉系统的衍射很弱，这项研究必须在同步辐射光源上进行。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. All X-ray experiments proposed will, whenever revealing, be matched by parallel EM experiments on 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 continue our novel recent work on crossbridge braking actions. The purpose of this rapid access proposal is to evaluate freezing conditions that can enhance sample lifetime so that much higher resolution diffraction patterns 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 synchrtrron source. As far as we know this has not been successfully achieved in muscle before and may be expected to lead to novel findings.