Mass Mapping Of Macromolecular Assemblies

扫描透射电子显微镜（STEM）提供了一个通用的方法来确定分子质量，从而在大的蛋白质组装的亚基的安排。大分子被吸附到一个薄的支持膜和纳米尺寸的电子探针扫描整个试样，而收集的弹性散射信号。所得到的数字图像强度与样本的局部质量密度成比例。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。我们已经使用这种方法来确定螺旋体柑橘，这是蛋白质组装，在这个简单的细菌产生运动的细胞骨架的层次和空间组织。结构单位似乎是一个原纤维，约5 nm宽，由59 kDa蛋白质的二聚体组成;每个条带由7个原纤维对组装而成。完整条带的功能单元是一对对齐的原纤维，沿着该原纤维沿着，成对的二聚体形成四聚环状重复。平均而言，分离和纯化的条带含有14个原纤维或7个排列良好的原纤维对，这是在完整细胞中观察到的相同结构。

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 determine the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri, which is protein assembly that produces motility in this simple bacterium. The structural unit appears to be a fibril, ~ 5 nm wide, composed of dimers of a 59 kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell.