STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的目标是表征分子的形状、分子质量 单个大分子的分布和元素组成 大分子组装。这种组件对许多电池来说是关键 功能，它们的体外行为反映了它们的功能和 在完整细胞中的调节。这个项目依赖于一种独特的仪器 一种低温、高分辨率的场发射扫描传输 电子显微镜(STEM)用于分子量图谱和化学 DIRECT的平行电子能量损失谱(PEELS)分析 直接冷冻组织的冷冻薄膜和超薄冰冻切片。 鱿鱼脑运动蛋白的暗~场分子量图显示 这种马达蛋白的新构象，其中运动蛋白轻链 末端折叠回到分子铰链附近的茎上；这 导致柄区域明显缩短。潜力 这种构象的意义是通过对其进行质量分析而提出的 Kinesin~微管复合体，我们在那里发现单一的Kinesin 可以跨越典型间隔为25 nm的微管对， 仅与激动素的缩短构象一致。一种能力 从单个动蛋白到交叉桥微管意味着第二个， 先前未识别的轻链末端的微管结合部位 提示激动素可能在血管紧张素系统的稳定中发挥作用。 微管排列和微管滑动。我们已经应用了一种新的 一种基于低剂量剥离价电子区分析的方法 确定冻结层最佳厚度的冻结~水化剖面图 果皮元素分析及分布测量用冰冻切片 浦肯野细胞树突内的水。该方法与 高剂量皮尔斯光谱成像已被用来测量钙离子。 蒲肯野树枝状，灵敏度提高四倍。

The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of individual macromolecules and macromolecular assemblies. Such assemblies are critical to many cell functions, and their behavior in vitro reflects their function and regulation in intact cells. This project depends on a unique instrument a low~temperature, high~resolution, field~emission scanning transmission electron microscope (STEM) for molecular weight mapping and chemical analysis by parallel electron energy loss spectroscopy (PEELS) of directly frozen thin films and ultrathin cryo~sections of directly frozen tissues. Dark~field molecular weight mapping of squid brain kinesin has revealed a new conformation of this motor protein, in which the kinesin light chain end is folded back onto the stalk of the molecule near its hinge; this results in an apparently shortened stalk region. The potential significance of this conformation is suggested by mass analysis of kinesin~microtubule complexes, where we have found that single kinesins can crossbridge microtubule pairs with a typical spacing of 25 nm, consistent only with the shortened conformation of kinesin. The ability of single kinesins to crossbridge microtubules implies a second, previously unrecognized, microtubule binding site on the light~chain end of kinesin, and suggests that kinesin may play a role in stabilization of micro~tubule arrays and in microtubule sliding. We have applied a new method based on analyzing the valence electron region of a low~dose PEELS map of frozen~hydrated sections to determine the optimal thickness of cryosections for PEELS elemental analysis and to measure the distribution of water within Purkinje cell dendrites. This method in combination with high~dose PEELS spectrum imaging has been used to measure calcium in Purkinje dendrites with a fourfold improvement in sensitivity.