Mass Mapping Of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 7319038
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 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/7319038
• 关键词: Mass; Mapping; Macromolecular; Assemblies

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. We have implemented STEM mass mapping in a 300 kV transmission electron microscope (FEI Tecnai TF30) equipped with a field-emission source and probe forming electron optics. A high-efficiency annular dark-field detector was installed in a port after the final projector lens of the microscope column. Addition of this capability to an advanced electron microscope will enable cryo-electron microscopy of frozen hydrated specimens, which can be subsequently dehydrated in situ and imaged by STEM to determine the mass distribution within the same structures. By adjusting the microscope?s projector lenses, it is possible to vary the camera length so that the range of scattering angles selected by the annular detector can be optimized. We have shown that it is possible to quantify the mass measurements by incorporating a theoretical model for the scattering cross sections (recent NIST Database). 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 successfully tested the mass mapping system on the 300 kV FEI Tecnai electron microscope by analyzing the well known structures, tobacco mosaic virus particles and mollusk hemocyanin. We are now able to perform mass mapping with our 300 kV instrument as well as with our dedicated 100 kV VG Microscopes HB501 STEM. We have applied the technique to a number of projects including characterization of proteins associated with motility of the simple bacterium, Spiroplasma, and to determine the molecular mass of postsynaptic densities.

扫描透射电子显微镜（STEM）提供了一种多功能方法，用于确定分子质量，从而在大蛋白质组件中的亚基排列。我们已经在配备了现场发射源和探针形成电子光学的300 kV传输电子显微镜（FEI Tecnai TF30）中实现了茎质量映射。在显微镜柱的最终投影镜头之后，将高效的环形探测器安装在端口中。在晚期电子显微镜中添加这种能力将使冷冻水合样品的冷冻电子显微镜能够进行冷冻电子显微镜，随后可以将其原位脱水并通过stem成像以确定相同结构内的质量分布。通过调整显微镜的投影镜镜头，可以更改相机长度，以便可以优化由环形检测器选择的散射角度范围。我们已经证明，可以通过合并散射横截面（最近的NIST数据库）的理论模型来量化质量测量值。大分子被吸附到薄的支撑膜上，并在收集弹性散射信号的同时扫描了纳米尺寸的电子探针。所得的数字图像强度与试样的局部质量密度成正比。图像可以在低电子剂量下记录，而不会对感兴趣的结构进行重大辐射损害。我们通过分析众所周知的结构，烟草病毒颗粒和软体动物血质蛋白，成功地在300 kV Fei tecnai电子显微镜上测试了质量映射系统。 现在，我们能够使用300 kV仪器以及我们专用的100 kV VG显微镜HB501茎进行质量映射。我们将该技术应用于许多项目，包括与简单细菌，螺旋质的运动性相关的蛋白质的表征，并确定突触后密度的分子质量。