Optimizing cryo-EM structural analysis of G_i-coupling receptors via engineered G_t and Nb35 application

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-G_i complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-G_i complexes remain both challenging and resource-intensive. By employing eGα_t, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGα_t complex was previously reported. Using this modified G protein, we determined the structure of the ET_B-eG_t complex bound to the modified Nb35. The determined structure of ET_B receptor was the same as the previously reported ET_B-G_i complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-G_i complexes.