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) 通过准确测定具有任意形状和大小的大型蛋白质组装体的分子质量，为结构生物学做出了重大贡献。 尽管如此，STEM 质量测绘在极少数实验室中得到实施，其中大多数实验室都采用在 100 kV 及更低加速电压下运行的冷场发射枪 (FEG) 电子源。 在这里，我们展示了配备热辅助肖特基 FEG 的 300 kV 商用透射电子显微镜 (TEM) 也可以提供精确的 STEM 质量测量。 使用 NIST 数据库中的弹性散射横截面，我们表明测量的烟草花叶病毒和帽贝血蓝蛋白二聚体的绝对质量值与已知值的吻合程度优于 10%。 我们发现血蓝蛋白组装体的测量分子量与已知值的吻合度在 3% 以内。 在中间电压 (200-400 kV) 下运行的 FEG TEM 正在成为确定冷冻水合蛋白质组装体结构的常用工具。 使用同一仪器进行质量测定的能力可以提供有关组成正在研究其结构的蛋白质组装体的亚基数量的重要补充信息。