Acquisition of a State-of-the-Art Electron Cryomicroscope

9977556摘要该项目涉及购买和安装300 kEV场发射枪（FEG）透射电子显微镜（TEM）以确定细胞机器的结构。 细胞机器，如肌动蛋白细胞骨架，通常是很大的结构，难以研究的方法以外的电子冷冻显微镜。 例如，它已经有可能对接原子模型的细胞骨架的组成部分获得的X射线晶体到分子图的细胞骨架复合物获得的电子低温显微镜。 在许多情况下，丝状结构的分析已经扩展到10 A分辨率。 最先进的显微镜对这些研究至关重要。 在本项目期间获得和安装的显微镜将用于研究剪接体、电压门控离子通道、肠微绒毛的肌动蛋白束、细菌鞭毛马达和细丝、细菌气泡、受体激酶信号复合物以及肌动蛋白与肌球蛋白的复合物。 这些研究的目的是结构的原子模型。 在某些情况下，仅使用电子低温显微镜将获得原子分辨率的结构。 在其他情况下，通过X射线晶体学获得的原子模型将对接到通过低温显微镜获得的分子图中。 300 kEV FEG TEM对这些项目至关重要，因为显微照片的质量限制了地图的分辨率，就像物镜透镜的质量限制了光学显微镜的分辨率一样。 无论多么复杂，计算机处理都无法提取图像中不存在的内容。 在这方面，该显微镜的两个关键特征是更高的电压和FEG。 利用这些，可以拍摄高度散焦的图像，其在低分辨率下具有强对比度，但保留高分辨率细节。 低分辨率下的强对比度对于在平均之前精确对齐图像至关重要。 如果对齐（或失真校正）不准确，则会对非等效特征进行平均，分辨率将丢失。 这种显微镜将扩展当前地图的分辨率，因此，无论是直接还是通过将原子模型对接到分子地图中，都能够获得精确的原子模型。低温显微镜对培养年轻科学家至关重要。 由于所使用的显微镜代表了细胞机器结构研究的前沿，因此学生和博士后研究员必须接受这些技术的培训。 由于低温电子显微镜的能力已经得到证实，对这类科学家的需求正在迅速增加。 目前很少有地方配备这种培训。

9977556Abstract This project involves the acquisition and installation of a 300 kEV field emission gun (FEG) transmission electron microscope (TEM) to determine the structures of cellular machinery. Cellular machines such as the actin cytoskeleton are often large structures difficult to study by methods other than electron cryomicroscopy. For example, it has been possible to dock atomic models for the cytoskeletal components obtained by x-ray crystallography into the molecular maps of the cytoskeletal complexes obtained by electron cryomicroscope. Analysis of filamentous structures has been extended to 10 A resolution in many cases. A state of the art microscope is essential for these studies. The microscope to be obtained and installed during this project will be utilized for the study of the splicesome, voltage-gated ion channels, the actin bundle of the intestinal microvillus, the bacterial flagellar motor and filament, the bacterial gas vesicle, the receptor-kinase signaling complex and the complex of actin with myosin. The aim of these studies are atomic models of the structures. In some instances the structure will be obtained to atomic resolution using electron cryomicroscopy alone. In other cases, atomic models obtained by x-ray crystallography will be docked into molecular maps obtained by cryomicroscopy. The 300 kEV FEG TEM is essential to these projects because the quality of the micrographs limits the resolution of the map in the same way that the quality of the objective lens limits the resolution of the light microscope. No matter how sophisticated, computer processing cannot extract what is not present in the images. The two key features of this microscope in this regard are the higher voltage and the FEG. With these, it is possible to take highly defocused images which have strong contrast at low resolution but which preserve high resolution detail. The strong contrast at low resolution is essential for accurate alignment of images prior to averaging. If the alignment (or correction for distortion) is not accurate, one will average non-equivalent features and resolution will be lost. This microscope will extend the resolution of current maps and therefore, the ability to obtain accurate atomic models whether it be directly or by the docking of atomic models into molecular maps. The cryomicroscope is essential to the training of young scientists. Since the kind of microscopy being used represents a frontier in structural studies of cellular machinery, students and post doctoral fellows must be trained in these techniques. The need for such scientists is increasing rapidly now that the power of cryoelectron microscopy has been demonstrated. There are currently few places equipped for this kind of training.