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 特定个体的分布和元素组成 大分子和大分子组装。这样的集会是 对许多细胞功能至关重要，它们在体外的行为反映了 它们在完整细胞中的功能和调节。这个项目依赖于 一种独特的仪器--低温、高分辨率、场发射 扫描电子显微镜(STEM)--测定分子量 平行电子能量损失测绘和化学分析 直接冷冻薄膜和超薄膜的光谱(EELS) 直接冰冻组织的冰冻切片。暗~场分子量 来自鱿鱼轴浆的天然神经细丝的定位表明 鱿鱼的单位长度质量为38=B15 kDa/nm，类似于 为其他核素装配件报告的值。第二~差值鳗鱼光谱 结果表明，在核因子的10~nm区域内有30个P原子， 对应于KSP上可用的KSP位点的完全磷酸化 这些细丝的重亚单位。这些结果表明，鳗鱼是 能够检测出生理上相关的差异 磷酸化状态的有效空间分辨率为10~20 nm。我们 我应用了一种新的方法，基于分析价电子区 冰冻~水化切片的低剂量鳗鱼图谱以确定 水在小鼠肝脏和小脑皮质内的分布 已经经历了创伤引起的细胞肿胀。此方法在 结合高剂量鳗鱼光谱成像有望揭示 与创伤有关的水和钙分布的变化 亚细胞水平。

The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of specific 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 (EELS) of directly frozen thin films and ultrathin cryosections of directly frozen tissues. Dark~field molecular weight mapping of native neurofilaments (NFs) from squid axoplasm indicated that the mass per unit length of squid NFs was 38=B15 kDa/nm, similar to values reported for other NF assemblies. Second~difference EELS spectra revealed that there are 30 P atoms within the 10~nm region of the NF, corresponding to complete phosphorylation of available KSP sites on the heavy subunit of these filaments. These results indicate that EELS is capable of detecting physiologically relevant differences in phosphorylation states at a useful spatial resolution of 10~20 nm. We have applied a new method~based on analyzing the valence electron region of a low~dose EELS map of frozen~hydrated sections to determine the distribution of water within mouse liver and within cerebellar cortex that had undergone trauma~induced cellular swelling. This method in combination with high~dose EELS spectrum imaging is expected to reveal trauma~related changes in water and calcium distributions at the subcellular level.