![]() X-ray crystallography initially provided a robust method to determine many GPCR structures. ![]() Continued structural interrogation of GPCRs in all functional states, including unliganded apo, inactivated by antagonists, activated by agonists, and in complexes with downstream signaling proteins, remains critically important in understanding GPCR signal transduction as well as facilitating therapeutic development. Over the past decade, methodological advances have provided deep insights into the structure and mechanism of GPCR function. As a family, GPCRs represent an important class of drug targets for many human diseases 3. Upon agonist binding, GPCRs activate a wide variety of downstream signaling pathways by interacting with heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs), and arrestins 2. All GPCRs share a common seven-transmembrane helices (7TM) architecture and are divided into six families, from A to F 1. G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins. Here, we address this shortcoming by exploring different fusion protein designs, which lead to structures of antagonist bound A 2A adenosine receptor at 3.4 Å resolution and unliganded Smoothened at 3.7 Å resolution. Although a similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins.
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