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表征从大鼠前脑分离、快速冷冻并支撑在薄层碳基质上的突触后密度(PSD)的组成。PSD是一个复杂的分子机器，位于突触后膜下，负责组织突触后活动区的受体和信号分子。研究发现，当神经元暴露在缺血和其他兴奋条件下时，PSD会变厚，已知CaMKII酶参与了这种增厚。我们已经使用STEM质量映射来估计在兴奋条件下添加到PSD中的CaMKII分子的数量。对PSD总质量的了解为定量和化学计量学分析PSD的蛋白质组成的功能变化开辟了道路

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 characterize the composition of post-synaptic densities (PSDs) that are isolated from rat forebrain, frozen rapidly, and supported on thin carbon substrates. The PSD is a complex molecular machine, which underlies the post-synaptic membrane and is responsible for organizing receptors and signaling molecules at the post-synaptic active zone. It is found that PSDs become thicker upon exposure of the neurons to ischemia as well as other excitatory conditions and it is known that the enzyme CaMKII contributes to this thickening. We have used STEM mass mapping to estimate the number of CaMKII molecules that are added to the PSD under excitatory conditions. Knowledge of the total mass of the PSD opens the way to a quantitative and stochiometric analysis of functional changes in the protein composition of PSDs