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。我们使用这种方法来研究柑橘螺旋体的组成，这是一种简单的细菌，它因其产生运动的收缩细胞骨架而引起人们的兴趣，该细胞骨架也起到线性马达的作用。这个马达可以作为一个模型系统来理解细胞骨架在其他细胞中的功能。此外，为了确定收缩直线马达的组织结构，我们现在正在表征膜结构，并从蛋白质、脂肪和核酸含量方面确定螺旋体的组成。预计结果将提供一些关于维持细胞自由生活状态的最低要求的信息。

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 investigate the composition of Spiroplasma citri, a simple bacterium, which is of interest because of its motility-producing contractile cytoskeleton, which also functions as a linear motor. This motor may serve as a model system for understanding the function of the cytoskeleton in other cells. In addition, to determining the organization of the contractile linear motor, we are now characterizing the membrane structure and determining Spiroplasma's composition in terms of protein, lipid and nucleic acid content. It is expected that the results will provide some information about the minimum requirements to sustain the cell's free-living state.