Acquisition of a high resolution, high throughput cryo-electron microscope

项目摘要 冷冻电子显微镜（cryo-EM）是一种强大的技术，用于确定结构的 大分子复合物从冷冻水化标本，绕过限制强加的其他 结构生物学技术，如X射线晶体学和核磁共振。冷冻EM依靠成像个人 分子在高放大率，然后平均在一起的图像，数十万份的 三维的分子。Cryo-EM在结构生物学方法中是独一无二的， 能够从混合的分子群体中分辨出不同的构象状态， 在接近生理条件下研究柔性和异质大分子。 冷冻EM的最新技术进步使可实现的 分辨率，近原子分辨率的结构成为常规。这意味着冷冻电镜技术 与X射线晶体学在结构的精确度上相媲美， 可以进行高分辨率结构分析的大分子类型。这些 一些研究人员发现， 放弃其他结构技术批发和其他急于采用新技术， 这是对现有方法的补充。两项技术发展推动了这一转变： 一代电子显微镜设计的独特限制冷冻EM与改进的 光学和更高的吞吐量，以及先进的直接电子探测器，大大提高了 记录图像的分辨率。华盛顿大学已经在直接探测器技术上进行了投资， 申请资金购买高性能低温电子显微镜FEI Talos Arctica。 Talos Arctica是一款高性能的电子显微镜， 用于冷冻电镜。改进包括一个恒定功率的物镜透镜更大的热稳定性 和减少的滞后，用于自动化和无污染样品转移的低温自动加载器， 更好的真空系统，用于保持样品不受污染， 机械精度和稳定性，以及一个封闭的平台，用于更好的环境控制， 稳定在Talos Arctica中体现的改进的净效果是增加了 获取更高质量的数据。显微镜的质量、可靠性和吞吐量将扩大 为校园和该地区的用户提供cryo-EM。这台显微镜可以 新的研究路线，并补充正在进行的研究计划，在不同的领域，如基础 细胞生物学和生物化学，传染病和疫苗开发，膜蛋白 结构、细胞应激反应、代谢、神经生物学和癌症。

Project Abstract Cryo-electron microscopy (cryo-EM) is a powerful technique for determining the structures of macromolecular complexes from frozen hydrated specimens, bypassing constraints imposed by other structural biology techniques like X-ray crystallography and NMR. Cryo-EM relies on imaging individual molecules at high magnification, then averaging together images of hundreds of thousands of copies of the molecule in three dimensions. Cryo-EM is unique among structural biology approaches in being able to resolve different conformational states from a mixed population of molecules, making it ideal to study flexible and heterogeneous macromolecules under near-physiological conditions. Recent technological advances in cryo-EM have generated a quantum leap in achievable resolution, with near-atomic resolution structures becoming routine. This means that cryo-EM has come to rival X-ray crystallography in the accuracy of structures that can be determined, as well as expanding the types of macromolecules that can be subjected to high-resolution structural analysis. These advances have created tectonic shifts in the landscape of structural biology, with some investigators abandoning other structural techniques wholesale and others rushing to adopt the new technologies as a compliment to existing approaches. Two technological developments have driven this shift: a new generation of electron microscopes designed for the unique constraints of cryo-EM with improved optics and higher throughput, and advanced direct electron detectors that dramatically improve the resolution of recorded images. UW has already invested in direct detector technology, and here we are requesting funds to acquire a high performance cryo-electron microscope, the FEI Talos Arctica. The Talos Arctica is a high performance electron microscope designed from the ground up to be used for cryo-EM. Improvements include a constant power objective lens for greater thermal stability and reduced hysteresis, a cryo-autoloader for automated and contamination free specimen transfer, better vacuum system for maintaining specimens without contamination, a piezo stage for improved mechanical precision and stability, and an enclosed platform for better environmental control and stability. The net effect of the improvements embodied in the Talos Arctica is an increased rate of acquisition of higher quality data. The quality, reliability, and throughput of the microscope will broaden the accessibility of cryo-EM for users across campus and in the region. This microscope will enable new lines of research and complement ongoing research programs in fields as diverse as fundamental cell biology and biochemistry, infectious diseases and vaccine development, membrane protein structure, cellular stress responses, metabolism, neurobiology, and cancer.