300 kV transmission electron microscope for cryo applications

在所有的项目中，一个关键的方面是实现与设想的仪器是大的，灵活的，多因素的生物大分子和超分子复合物的三维结构的测定。这些复合物的大尺寸、柔性性质和低稳定性限制了通过X射线晶体学或NMR确定其结构的可能性，使得单颗粒cryo-EM成为唯一可行的替代方案。需要近原子分辨率的cryo-EM图（即约3.5 μ m或更好）来从头模拟结构，对这些结构中不包含在组分亚复合物的可用高分辨率结构中的部分进行建模，或准确地对不同的构象状态进行建模。要进入这些分辨率制度，需要一个300 kV的冷冻EM仪器与相位板，能量过滤器和直接电子检测器。此外，需要自动化数据收集的能力，以使大量的显微照片的采集-只有足够的颗粒图像，随后的构象分选将提供足够的图像，在每个类别的高分辨率结构分析的所有构象状态。这样的结构将允许产生关于所涉及的分子机制的分子机制或调节模式的详细假设，并且将为设计干扰突变以直接测试推定的机制提供基础。

A key aspect in all of the projects to be realized with the envisaged instrument is the determination of threedimensional structures of large, flexible, multi -factorial biomacromolecular and supramolecular complexes. The large size, flexible nature and low stability of these complexes limit possibilities to determine their structures by X-ray crystallography or NMR, leaving single particle cryo-EM as the only viable alternative. Near-atomic resolution cryo-EM maps (i.e. around 3.5 Å or better) are required to model the structures de novo, to model the portions of these structures that are not contained in available high-resolution structures of component subcomplexes or to accurately model different conformational states. To enter these resolution regimes, a 300 kV cryo-EM instrument with phase plate, energy filter and direct electron detector is required. Additionally, a capability for automated data collection is required to enable acquisition of a large number of micrographs - only with enough particle images, subsequent conformational sorting will provide enough images in each class for high-resolution structure analysis of all conformational states. Such structures would allow give rise to detailed hypotheses regarding the molecular mechanisms or modes of regulation of the molecular machineries involved, and would provide a basis for designing interfering mutations to directly test the putative mechanisms.