STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

本项目的目标是在体外表征的形状，分子 特定个体的重量分布和元素组成 大分子和大分子组装，重点是组件 的细胞骨架。这个项目依赖于一个独特的工具-一个 场发射扫描透射电子显微镜（STEM）-- 配备暗场检测器（用于绘制分子量 分子分布在2nm的空间分辨率）和平行 电子能量损失（EELS）光谱仪（用于检测生理 磷酸化状态的相关差异，分辨率为10-20 nm）。 将此方法应用于快速冷冻、冻干的神经丝 从乌贼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/ 长度为22.7 ± 0.8 kDa/nm，这意味着只有8个卷曲的大肠杆菌 二聚体/横截面。EELS结果表明，每个重链有四条重链。 横截面，每个含有约50至60磷酸盐;这 基本上是氨基酸序列预测的最大值。我们有 也开始利用能量过滤电子显微镜（EFTEM）， 细胞磷分析的补充方法。 EFTEM 已被用于绘制磷分布图， 分子组装，但在高电子剂量，这导致标本 损害 最近的实验表明， 直接冷冻薄膜的定量，元素特异性图像， 低剂量下的生物样本，因此可能在高剂量下 分辨率因此，我们获得了磷特异性低剂量图像 单纯疱疹病毒颗粒的DNA核心。的 这些图像之间的差异显示了统计学显著性 病毒内DNA分布的定量图。 这些 结果表明，进行这样的实验是可能的。 即使在完全水合的样品中。

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.