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).