Mass Mapping of Macromolecular Assemblies

近四十年来，扫描透射式电子显微镜(STEM)为结构生物学做出了重大贡献，它提供了任意形状和大小的大型蛋白质组件的精确分子质量测定。尽管如此，极少数实验室已经实施了STEM质量映射，其中大多数实验室使用了工作在100千伏及以下的加速电压下的冷场发射枪(FEG)电子源。在这里，我们展示了配备热辅助Shottky FEG的300千伏商用透射电子显微镜(TEM)也可以提供准确的STEM质量测量。利用NIST数据库中的弹性散射截面，我们发现烟草花叶病毒和血蓝蛋白二聚体的绝对质量测量值与已知值在10%以内。我们发现，测得的血蓝蛋白组装体的相对分子质量与已知值在3%以内。在中等电压(200-400千伏)下工作的FEG TEM正在成为确定冷冻水合蛋白组件结构的常用工具。用同一仪器进行质量测定的能力可以提供关于组成其结构被研究的蛋白质组件的亚基数量的重要补充信息。

For almost four decades, the scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold-field emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can also provide accurate STEM mass measurements. Using the elastic-scattering cross sections from the NIST database, we show that the measured absolute mass values for tobacco mosaic virus and limpet hemocyanin didecamers agree with the known values to within better than 10%. We find that the measured molecular weight of the hemocyanin assemblies agrees with the known value to within 3%. FEG TEMs operating at intermediate voltages (200-400 kV) are becoming common tools for determining the structure of frozen hydrated protein assemblies. The ability to perform mass determination with the same instrument can provide important complementary information about the numbers of subunits comprising the protein assemblies whose structures are being studied.