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bioRxiv · 10.1101/2023.10.07.561347

Optimizing Cryo-EM Structural Analysis of Gi-coupling Receptors via Engineered Gt and Nb35 Application

Oshima, H. S.·Sano, F. K.·Akasaka, H.·Iwama, A.·Shihoya, W.·Nureki, O.
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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-Gi 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-Gi complexes remain both challenging and resource-intensive. By employing eGt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes. HighlightsO_LIThe study introduces the engineered G protein subunit eGT, which enhances the resolution of GPCR-G protein structures by suppressing G protein conformational fluctuations and is particularly beneficial for Gi-coupled receptors. C_LIO_LIThe cryo-EM structure of the ETB receptor complexed with eGt-Nb35 reveals improved map quality, reduced anisotropy, and isotropic density distribution, increasing the accuracy of structural analysis. C_LIO_LIStructural comparison between ETB-Gi and ETB-eGt reveals similar receptor-G protein interactions, demonstrating the utility of eGt-Nb35 for studying GPCR-Gi complexes and the potential for broader applications within the Gi family. C_LI

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Oshima, H. S., Sano, F. K., Akasaka, H., Iwama, A., Shihoya, W., Nureki, O.. 2023-10-07. Optimizing Cryo-EM Structural Analysis of Gi-coupling Receptors via Engineered Gt and Nb35 Application. https://doi.org/10.1101/2023.10.07.561347

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