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

这个项目包括购置和安装一台300keV的场发射枪(FEG)透射电子显微镜(TEM)，以确定细胞机械的结构。细胞机器，如肌动蛋白细胞骨架，通常是很难用电子冷冻显微镜以外的方法研究的大型结构。例如，通过X射线结晶学获得的细胞骨架成分的原子模型与通过电子冷冻显微镜获得的细胞骨架复合体的分子图谱对接是可能的。在许多情况下，对丝状结构的分析已扩展到10A分辨率。对于这些研究来说，最先进的显微镜是必不可少的。在该项目中获得和安装的显微镜将用于研究剪接的电压门控离子通道、肠道微绒毛的肌动蛋白束、细菌鞭毛马达和细丝、细菌气泡、受体-激酶信号复合体以及肌动蛋白与肌球蛋白的复合体。这些研究的目的是建立结构的原子模型。在某些情况下，仅使用电子冷冻显微镜就可以获得原子分辨率的结构。在其他情况下，通过X射线结晶学获得的原子模型将与通过冷冻显微镜获得的分子图对接。300keV的FEG电子显微镜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东西。在这方面，这款显微镜的两个关键特点是较高的电压和FEG。有了这些，就有可能拍摄高度散焦的图像，这些图像在低分辨率下具有强烈的对比度，但保留了高分辨率的细节。在低分辨率下的强对比度对于在平均之前准确对齐图像是必不可少的。如果对齐(或失真校正)不准确，将平均不等价的特征，分辨率将丢失。这种显微镜将扩展当前地图的分辨率，因此，无论是直接获得准确的原子模型，还是通过将原子模型对接到分子地图中，都可以获得准确的原子模型。冷冻显微镜对年轻科学家的培训是必不可少的。由于所使用的显微镜是细胞机械结构研究的前沿，学生和博士后研究员必须接受这些技术方面的培训。由于低温电子显微镜的力量已经被证明，对这样的科学家的需求正在迅速增加。目前，配备这种培训的场所很少。

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.