STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

这个项目的目标是在体外表征分子的形状、分子 特定个体的体重分布和元素组成 大分子和大分子组装，重点放在组件上 细胞骨架的。这个项目依赖于一种独特的工具--一种 场发射扫描电子显微镜(STEM) 配备暗场探测器(用于绘制分子量图 空间分辨率为2 nm的分子的分布)和平行的 电子能量损失(EELS)光谱仪(用于生理检测 分辨率为10-20的磷酸化状态的相关差异 NM)。将这种方法应用于速冻、冷冻干燥的神经细丝 从鱿鱼巨大的轴突中分离出来，我们已经能够推导出一种新的 重链排列的结构模型(高度 天然轴突中的轻链(弱磷酸化)和轻链(弱磷酸化) 神经丝。STEM测量给出了神经丝质量-每- 22.7加上负0.8 kDa/nm的长度，这意味着只有8个卷曲的 每横截面的二聚体。鳗鱼的结果显示每个人有四条重链 横截面，每个含有大约50到60个磷酸盐；这 基本上是氨基酸序列预测的最大值。我们有 也开始开发能量过滤电子显微镜(EFTEM)作为 细胞磷分析的一种补充方法。EFTEM 以前曾被用来绘制磷的分布图 分子组装，但在高电子剂量下，这将导致样品 损坏。最近的实验表明，有可能获得 直接冷冻的薄膜的定量、元素特定图像 低剂量的生物标本，因此可能处于高剂量 决议。因此，我们获得了磷特定的、低剂量的图像 带有和不带有DNA核心的单纯疱疹病毒颗粒。这个 这些图像之间的差异揭示了统计上的显著差异 DNA在病毒内的分布的定量地图。这些 结果表明，进行这样的实验是可能的。 即使是在完全水合的标本中。

The goal of this project is to characterize in vitro the shape, molecular weight distribution and elemental composition of specific individual macromolecules and macromolecular assemblies, with emphasis on components of the cytoskeleton. This project depends on a unique instrument -- a field-emission scanning transmission electron microscope (STEM) -- equipped with dark-field detectors (for mapping the molecular weight distribution of molecules at a spatial resolution of 2 nm) and a parallel electron energy loss (EELS) spectrometer (for detecting physiologically relevant differences in phosphorylation states at a resolution of 10-20 nm). Applying this method to rapidly-frozen, freeze-dried neurofilaments isolated from the squid giant axon, we have been able to derive a novel structural model for the arrangement of heavy chains (highly phosphorylated) and light chains (weakly phosphorylated) in native axonal neuro-filaments. The STEM measurements give a neurofilament mass-per- length of 22.7 plus minus 0.8 kDa/nm which implies only eight coiled-coli dimers per cross-section. The EELS results indicate four heavy chains per cross section, each containing approximately 50 to 60 phosphates; this is essentially the maximum predicted by the amino acid sequence. We have also begun to exploit an energy-filtering electron microscope (EFTEM) as a complementary approach for the analysis of cellular phosphorus. EFTEM has been previously used for mapping of phosphorus distributions in molecular assemblies, but at high electron dose, which leads to specimen damage. Recent experiments now show that it is possible to obtain quantitative, element-specific images of directly frozen thin films of biological specimens at low dose and therefore potentially at high resolution. Thus, we have obtained phosphorus-specific, low-dose images of herpes simplex virus particles with and without their DNA cores. The difference between these images reveals a statistically significant quantitative map of the distribution of the DNA within the virus. These results suggest that it may be possible to carry out such experiments even in fully hydrated specimens.