Optimizing Cryo-EM Structural Analysis of G i -coupling Receptors via Engineered G t and Nb35 Application
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Abstract
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.
Highlights
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The study introduces the engineered G protein subunit eGα T , which enhances the resolution of GPCR-G protein structures by suppressing G protein conformational fluctuations and is particularly beneficial for G i -coupled receptors.
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The cryo-EM structure of the ET B receptor complexed with eG t -Nb35 reveals improved map quality, reduced anisotropy, and isotropic density distribution, increasing the accuracy of structural analysis.
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Structural comparison between ET B -G i and ET B -eG t reveals similar receptor-G protein interactions, demonstrating the utility of eG t -Nb35 for studying GPCR-G i complexes and the potential for broader applications within the G i family.
