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 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 complexes remain both challenging and resource-intensive. By employing eGα, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGα complex was previously reported. Using this modified G protein, we determined the structure of the ET-eG complex bound to the modified Nb35. The determined structure of ET receptor was the same as the previously reported ET-G complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-G complexes.
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