Abstract 1847 Capturing multiple conformations of glycyl radical enzymes found in the human gut microbiome using single particle cryoEM

Nature has devised an array of enzymatic cofactors that enable challenging chemical transformations to occur on a timescale compatible with biological life. Radical cofactors are commonly utilized to accomplish such chemistry-their high reactivity allows for transformations that would be inaccessible otherwise. Protein-based amino acid radicals are one example of a simple yet powerful radical cofactor capable of catalyzing diverse chemistry. The glycyl radical enzyme (GRE) superfamily is a prominent example of this type of cofactor and the catalytic power it possesses. Members of this strictly anaerobic enzyme family are commonly encoded by bacteria that reside within the human gut microbiome and play notable roles in human health including the metabolism of the abundant amino acid hydroxyproline found in collagen, formation of the disease-associated metabolite hydrogen sulfide, and production of trimethylamine (TMA) from choline, which has been linked to heart disease. Structural characterization of these enzymes provides insight into their catalytic mechanisms as well as avenues to control their activity as it is relevant in human health and disease. Here, we present the use of single-particle cryogenic electron microscopy (cryoEM) to capture various conformational states of glycyl radical enzymes from the human gut microbes Olsenella uli and Clostridium scatologenes. New computational tools such as CryoDRGN have enabled the sorting of heterogeneity within single EM datasets in silico, providing a powerful platform by which to capture multiple states of enzymes, and providing new insight into the range of enzyme conformations that may exist in a biological setting. With these tools, we were able to sort heterogeneous states from a single high-quality EM dataset. The Drennan Lab is funded by Howard Hughes Medical Institute and the National Institute of Health.