Acquisition of Equipment for Structural Studies of Macromolecular Assemblies Using Cryo-EM

项目摘要 R35资助下的实验研究主要集中在时间分辨（TR）冷冻电子显微镜 （cryo-EM）研究真核生物翻译过程中的短暂状态。TR cryo-EM是一种很有前途的新方法， 获得关于可以在体外进行的生物过程的动力学的信息。我们成功 这项技术在细菌翻译中的应用已经发表在《结构》、《自然》和《自然》杂志上。 通讯 这项研究需要大规模的高质量纯化核糖体和蛋白质因子--二十到五十个 比常规印迹冷冻EM实验中的样品量多10倍。 我们目前的AKTA净化系统老化，并显示出许多问题，这些问题在 本建议书的设备部分。我们无法使用核心设备或其他团体的设备， 既在可及范围内（即，在同一个校园）和RNA酶免费，一个严格的要求， 涉及核糖体。此外，制造商最近决定停止对该模型的支持 在不到一年的时间里。 因此，为了确保我们的研究不受干扰，我们要求补充设备，使我们能够 购买AKTA pure 25 M（Cytiva产品编号：29018226）纯化系统（122，412.70美元）。

PROJECT SUMMARY The experimental research under the parent R35 grant focuses on time-resolved (TR) cryo-electron microscopy (cryo-EM) to study short-lived states during translation in eukaryotes. TR cryo-EM is a promising new way to obtain information about the dynamics of biological processes that can be carried out in vitro. Our successful applications of this technology to translation in bacteria have been published in Structure, Nature and Nature Communications. This research requires large-scale high-quality purifications of ribosomes and protein factors -- twenty to fifty times more sample quantity than in normal blotting cryo-EM experiments. Our present AKTA purification system is aging and shows numerous problems, which are detailed in the Equipment section of this proposal. We do not have access to core equipment or other groups’ equipment that is both in reach (i.e., on the same campus) and RNAse-free, a stringent requirement for any preparations involving ribosomes. Moreover, the manufacturer has recently decided to discontinue support of this model within less than a year. Thus, to ensure uninterrupted pursuit of our research we request an equipment supplement allowing us to purchase an AKTA pure 25 M (Cytiva Product Number: 29018226) purification system ($122,412.70).