Native mass spectrometry prescreening of G protein-coupled receptor complexes for cryo-EM structure determination

TL;DR

A new method using mass spectrometry helps prepare GPCR samples for cryo-EM, enabling high-resolution structures with fewer data.

Contribution

Introduces an online buffer exchange-native MS method combined with Direct Mass Technology for efficient GPCR sample preparation.

Findings

The OBE-nMS+DMT method achieved a 3.51 Å resolution structure of the GPR119-Gs complex using only 396 cryo-EM movies.

The method enables high-throughput analysis and guides optimal sample conditions for cryo-EM studies of membrane proteins.

This approach improves sample preparation for unstable and dynamic GPCR complexes.

Abstract

G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryo-EM have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.