Calcineurin-fusion facilitates Cryo-EM Structure Determination of a Family A GPCR

Advances in singe-particle cryo-electron microscopy (cryo-EM) have made possible to solve the structures of numerous Family A and Family B G protein coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extra-membrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6 and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β₂ adrenergic receptor (β₂AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.