Myofibrillar Structure of Striated Muscle

描述(由申请人提供)：令人兴奋的最新进展有望从马达分子的时间分辨结构分析中获得继续的启示 在肌肉方面，阐明个人的运动动作以及它们的整合 整个肌肉的合奏表演。高准晶级数 巨型水虫的甘油昆虫飞行肌肉(IFM) 每种生理状态的独特X射线衍射(X射线)，并允许 肌球蛋白交叉桥的超薄切片电子显微镜(EM)图像。 图像载体与自然X射线模式的比较测量和验证 薄层电磁的保真度。新型同步辐射X射线光束线和探测器 允许使用新方法。我们将协调时间切片同步加速器X射线衍射 利用来自快速冷冻纤维的EM 3-D重建(3-D)来整合 机械和结构数据，用于肌球蛋白的毫秒级快照 力量冲刺。基于活动状态下的交叉桥分集的三维分析 关于三维电磁层析成像，它可以显示非平均结构，但也可以 允许部分或全部平均，以及3D对应分析 对不同的人行横桥形状进行分类和计数。 主动型、刚构型和悬索型IFM高力跨桥荷载诱导变形 AMPPNP状态将通过改变阶梯/坡道长度和 对冷冻纤维和化学固定纤维进行了X射线、3DEM研究，并进行了拟合。 肌球蛋白和肌动蛋白的晶体结构模型。部队生成计划将是 以这些配件为原型。低力(PowerStroke前)有源电桥，将 通过冷却、高(PI)和PI类似物(BDM、钒酸、 AlF4-，BeFx)，将与使用X射线、3D-EM和 原子模型的拟合。速冻IFM纤维的X射线低温衍射法 被开发来防止高通量辐射损伤，同时记录高 分辨率曝光，并冻结捕获稳定的，不断变化的，可能是动态的 各州。肌球蛋白在IFM交叉桥起始处的S1-S2连接将被探测 通过低盐晶格膨胀和高盐疏松粗丝 我的脊梁。弛豫IFM中的粗丝和A带结构将是 根据我们在20度、20度和40度(正常飞行)时的X射线数据协作建模 活体温度)，使用我们的辅助EM研究来生成模型 几何图形。

DESCRIPTION (provided by applicant): 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.