300 kV transmission electron microscope for cryo applications

在所有项目的一个关键方面，将实现与设想的仪器是确定三维结构的大，灵活的，多因子的生物大分子和超分子复合物。这些配合物的大尺寸，柔韧性和低稳定性限制了通过x射线晶体学或核磁共振确定其结构的可能性，使单颗粒低温电镜成为唯一可行的替代方案。需要近原子分辨率的低温电镜图（即大约3.5 Å或更好）来从头模拟结构，以模拟这些结构中不包含在组件亚配合物的可用高分辨率结构中的部分，或准确地模拟不同的构象状态。为了进入这些分辨率范围，需要一台300千伏的低温电镜仪器，配备相位板、能量滤波器和直接电子探测器。此外，需要自动收集数据的能力来获取大量的显微照片-只有有足够的粒子图像，随后的构象排序将在每个类别中提供足够的图像，用于所有构象状态的高分辨率结构分析。这样的结构将允许产生关于分子机制或所涉及的分子机制的调节模式的详细假设，并将为设计干扰突变以直接测试假定机制提供基础。

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.