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

项目摘要 低温电子显微镜(Cryo-EM)是一种确定结构的强有力的技术 来自冷冻水化样品的大分子复合体，绕过了其他 结构生物学技术，如X射线结晶学和核磁共振。CRYO-EM依靠个人成像 分子在高倍率下，然后将数十万个拷贝的图像平均在一起 三维的分子。冷冻-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中解析不同的构象状态，使其成为理想的 在接近生理的条件下研究柔性和非均相大分子。 低温EM的最新技术进步在可实现的 分辨率，近原子分辨率结构成为常规。这意味着冷冻-EM来了 在可以确定的结构的准确性上与X射线结晶学相媲美，以及在扩展 可以进行高分辨率结构分析的大分子的类型。这些 随着一些研究人员的研究，进步已经在结构生物学的版图上产生了构造变化 完全放弃其他结构技术，以及其他急于采用新技术的人，如 这是对现有方法的赞扬。两项技术发展推动了这一转变：一种新的 为低温电子显微镜的独特约束而设计的电子显微镜的产生与改进 光学和更高的吞吐量，以及先进的直接电子探测器，显著提高了 录制图像的分辨率。UW已经在直接探测器技术上进行了投资，现在我们来了 申请资金购买高性能冷冻电子显微镜FEI Talos Arctica。 Talos Arctica是一款全新设计的高性能电子显微镜 用于低温EM。改进包括恒倍物镜，以实现更高的热稳定性 和减少滞后，用于自动和无污染的样本转移的低温自动加载器， 更好的真空系统，保持样品不受污染，改进的压电台 机械精度和稳定性，以及更好的环境控制和封闭式平台 稳定性。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、可靠性和生产量将会扩大 整个校园和该地区的用户都可以使用Cryo-EM。这台显微镜将使 新的研究路线和对基础等不同领域正在进行的研究计划的补充 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、新陈代谢、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.