Mass Mapping Of Macromolecular Assemblies

扫描电子显微镜(STEM)提供了一种通用的方法来确定分子质量，从而确定大的蛋白质组装中亚单位的排列。大分子被吸附到一层薄的支撑膜上，在采集弹性散射信号的同时，一个纳米尺寸的电子探针在样品上扫描。由此产生的数字图像强度与样本的局部质量密度成正比。可以在低电子剂量下记录图像，而不会对感兴趣的结构造成显著的辐射损伤。我们使用这种方法测量了从简单细菌螺旋体中提取的膜的单位面积质量以及它的收缩细胞骨架，目的是建立一个对运动性研究感兴趣的细胞成分的清单。我们已经测试了在我们的100KV场发射STEM中扩展质量映射方法的可行性，通过成像十年期团簇，每个团簇包含11个金原子，吸附在一个薄的碳载体上。我们现在正在研究单马来酰亚胺十一聚体标记特定蛋白质的应用，这些蛋白质含有嵌入在膜中的游离巯基，这些蛋白质被十一聚体标记到脂类上。

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic-scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images can be recorded at low electron dose without significant radiation damage to the structures of interest. We have used this approach to measure mass per area of membranes extracted from the simple bacterium, Spiroplasma, as well as its contractile cytoskeleton, with the aim of constructing an inventory of the cellular components, which is of interest in the study of motility. We have tested the feasibility of extending the mass mapping approach in our 100 kV field-emission STEM by imaging undecagold clusters, each containing eleven gold atoms, adsorbed onto a thin carbon support. We are now investigating the application of monomaleimide undecagold to label specific proteins containing free sulfhydryl groups embedded in membranes that are labeled by undecagold attached to lipids.