300 kV transmission electron microscope for cryo applications

适用于低温应用的 300 kV 透射电子显微镜

• 批准号: 384108375
• 金额: --
• 依托单位: Freie Universität Berlin
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/384108375?language=en
• 关键词: 300; kV; transmission; electron; microscope

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.

利用设想的仪器实现的所有项目中的一个关键方面是确定大的、灵活的、多因素的生物大分子和超分子络合物的三维结构。这些络合物的尺寸大、性质灵活、稳定性低，限制了通过X射线结晶学或核磁共振确定其结构的可能性，使单颗粒低温EM成为唯一可行的替代方案。需要近原子分辨率的冷冻-EM图(即约3.5？或更高)来模拟从头开始的结构，模拟这些结构中未包含在现有高分辨率结构中的部分，或精确模拟不同的构象状态。要进入这些分辨率区域，需要一台带有相位板、能量过滤器和直接电子探测器的300千伏低温电磁仪。此外，为了能够获取大量的显微照片，需要具备自动数据收集的能力--只有在有足够的颗粒图像的情况下，后续的构象分类才能在每一类中提供足够的图像，以便对所有构象状态进行高分辨率结构分析。这样的结构将允许产生关于所涉及的分子机制或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的机制提供基础。