PROJECT SUMMARY/ABSTRACT The Apicomplexa phylum contains many parasites that infect and cause disease in humans and animals, including the agents that cause malaria, toxoplasmosis, and babesiosis. Babesia is a tick-borne intracellular parasite that causes severe to fatal diseases, with broadening geographic distribution because of climate change and increasing suburban developments in forested areas. There is no vaccine against human babesiosis, and the current therapies have limited efficacy. Considering the complex life cycle of Babesia species, their survival depends on the precise control of their gene expression, which is mostly regulated post-transcriptionally (e.g., during protein synthesis). Babesia species have a unique, non-photosynthetic organelle named apicoplast, derived from a secondary endosymbiotic event. In addition to having ribosomes in its cytoplasm and mitochondria, the apicoplast contains its own ribosomes. Little is known about the structure of these ribosomes, although they are thought to be highly simplified, with prokaryotic-like components. They are inhibited by antibacterial drugs that have some limited antiparasitic activity. The absence of a molecular structure of any apicoplast ribosomes limits the rational design of antibiotics with improved antiparasitic properties. In the first aim of this research proposal, we will establish a Babesia divergens strain expressing an hemagglutinin (HA)- tagged triose phosphate transporter on the outer membrane of the apicoplast. This strategy will allow the affinity purification of a high-quality organelle preparation, followed by the purification of apicoplast ribosomes. In the second aim, we will define the components of apicoplast ribosomes and determine their structure using cryo- electron microscopy (cryo-EM). Our goal is thus to elucidate the fundamental molecular machinery that apicomplexan parasites use to synthesize proteins in the highly specialized apicoplast organelle. Our work will lay the foundation for studies that interrogate these parasites' gene regulation mechanisms and help inspire the design of better antiparasitic agents.