Mass Mapping of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 7593811
• 负责人: Richard Leapman
• 金额: $ 1.59万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593811
• 关键词: Acceleration; Cryoelectron Microscopy; Databases; Electron Microscope; Electrons; Freezing; Goals; Guns; Hemocyanin; Laboratories; Maps; Measurement; Measures; Molecular Weight; Noise; Numbers; Proteins; Scanning; Scanning Transmission Electron Microscopy Procedures; Shapes; Signal Transduction; Source; Structural Biologist; Structure; Techniques; Tobacco Mosaic Virus; detector; instrument; interest; macromolecular assembly; molecular mass; research study; size; structural biology; tool; transmission process; voltage

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.

近四十年来，扫描透射电子显微镜（STEM）通过提供具有任意形状和大小的大型蛋白质组件的分子质量的精确测定，为结构生物学做出了重大贡献。尽管如此，STEM质量测绘已经在极少数实验室实施，其中大多数实验室使用的是在100 kV及以下加速电压下工作的冷场发射枪（FEG）电子源。在这里，我们证明了300 kV商用透射电子显微镜（TEM）配备热辅助Shottky FEG也可以提供精确的STEM质量测量。利用NIST数据库的弹性散射截面，我们发现烟草花叶病毒和帽贝血青素二十聚体的测量绝对质量值与已知值一致，在10%以内。我们发现血青素组装体的分子量与已知值在3%以内一致。在中压（200-400 kV）下工作的FEG tem正成为测定冷冻水合蛋白组装结构的常用工具。使用相同的仪器进行质量测定的能力可以提供关于组成所研究结构的蛋白质组合的亚基数量的重要补充信息。