Mass Mapping Of Macromolecular Assemblies

扫描透射电子显微镜（STEM）提供了一种通用的方法来确定分子质量，从而确定大蛋白质组装中亚基的排列。大分子被吸附到一层薄薄的支撑膜上，一个纳米大小的电子探针在样本上扫描，同时收集弹性散射信号。由此产生的数字图像强度是成比例的局部质量密度的标本。图像可以在低电子剂量下记录，而不会对感兴趣的结构造成明显的辐射损伤。我们已经用这种方法来确定柑橘螺旋体的细胞骨架的层次和空间组织，这是在这个简单的细菌中产生运动的蛋白质组装。该结构单元似乎是由59 kDa蛋白的二聚体组成的纤维，约5 nm宽；每条缎带都是由七对纤维组合而成。完整条带的功能单位是一对排列整齐的原纤维，沿着它们成对的二聚体形成四聚体环状重复。平均而言，分离和纯化的条带含有14根原纤维或7对排列良好的原纤维，这些原纤维与完整细胞中观察到的结构相同。

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.