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）通过准确地确定具有任意形状和大小的大蛋白质组件的分子质量，从而对结构生物学做出了重大贡献。 然而，很少有实验室实施了STEM质量映射，其中大多数使用冷场发射枪（FEG）电子源以100 kV和较低的加速电压运行。 在这里，我们表明，配备有热辅助的Shottky FEG的300 kV商业传输电子显微镜（TEM）也可以提供准确的茎质量测量值。 使用NIST数据库中的弹性散布横截面，我们表明，测得的烟草镶嵌病毒和lim虫血液糖蛋白Docamers的绝对质量值与已知值一致，超过10％以内。 我们发现，测得的血氨酸组件分子量与已知值一致，达到3％以内。 在中间电压（200-400 kV）上运行的FEG TEMS已成为确定冷冻水合蛋白质组件结构的常见工具。 使用相同仪器进行质量测定的能力可以提供有关包括研究结构的蛋白质组件的亚基数量的重要互补信息。