300 keV FEG Electron Cryo Microscope for Single Particle Analyses and Tomography

低温电子显微镜（cryo-EM）的最新技术发展导致了分辨率提高方面的重大突破，因此，在其适用性。使用新型直接电子探测器，冷冻EM的两个主要应用，单粒子分析和冷冻断层扫描，现在通常分别达到约3 μ m和10 μ m的分辨率。这使得单粒子cryo-EM成为结构生物学中通常由X射线晶体学解决的广泛问题的首选方法。在冷冻断层扫描的情况下，提高的分辨率允许在其天然细胞环境中识别单个大分子复合物，并且在子断层图像平均化之后，识别核酸和蛋白质二级结构。因此，这种发展导致了对数据收集时间的需求激增，特别是因为越来越小的颗粒以及天然的非丰富复合物可以成功地进行结构测定。然而，基因中心现有的设备无法满足这一需求：首先，一台高端显微镜的容量不足以满足日益增长的需求。其次，现有设备经过优化，可实现大颗粒的全自动高通量数据采集。因此，对于较小的颗粒或冷冻断层扫描，不能收集具有最佳质量的数据。因此，我们申请购买第二台高端冷冻显微镜，这通常会增加可用的数据收集选项和时间。更具体地说，拟议的显微镜计划配备一个相位板，一个能量过滤器和一个直接检测器，从而允许分析小颗粒以及冷冻断层扫描应用。

The recent technological developments in cryo-electron microscopy (cryo-EM) have led to a dramatic breakthrough with respect to improved resolution and, therefore, in its applicability. Using the novel direct electron detectors the two main applications of cryo-EM, single particle analysis and cryo-tomography, now routinely reach resolutions around 3 Å and 10 Å, respectively. This makes single particle cryo-EM the method of choice for a broad range of questions in structural biology that have usually been addressed by X-ray crystallography. In the case of cryo-tomography the improved resolution allows for the identification of individual macromolecular complexes in their native cellular environment and, after sub-tomogram averaging, the recognition of nucleic acid and protein secondary structure. As a result, this development has led to an explosion in the demand for data collection time, in particular, since increasingly smaller particles and also native, non-abundant complexes can be successfully subjected to structure determination. However, the currently available equipment at the Gene Center cannot satisfy this demand: firstly, the capacity of one high-end microscope is insufficient as to meet the increasing needs. Secondly, the existing equipment is optimized for fully automated high-through-put data collection of large particles. Consequently, data with optimal quality cannot be collected for smaller particles or for cryo-tomography. Therefore, we apply to purchase a second high-end cryo-microscope, which, in general, is expected to increase available data collection options and time. More specifically, the proposed microscope is planned to be equipped with a phase plate, an energy filter and a direct detector, thus allowing analysis of small particles as well as cryo-tomography applications.