Kim Donggyun, Liu Weijing, Viner Rosa, Cherezov Vadim
Bridge Institute, Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, CA 90089, USA; Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA.
Structure. 2024 Dec 5;32(12):2206-2219.e4. doi: 10.1016/j.str.2024.10.004. Epub 2024 Oct 28.
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 cryogenic electron microscopy (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-G 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.
G蛋白偶联受体（GPCRs）是重要的跨膜蛋白，在人类健康和疾病中发挥着关键作用。了解其原子水平的分子结构和构象状态对于推进药物开发至关重要。单颗粒低温电子显微镜（cryo-EM）的最新突破将GPCRs的结构生物学带入了一个新时代。然而，由于其稳定性差和高度动态的性质，制备适合cryo-EM分析的GPCR样品及其复合物仍然具有挑战性。在此，我们展示了我们结合直接质量技术（OBE-nMS+DMT）的在线缓冲液交换-天然质谱方法，该方法有助于高通量分析并指导样品制备。我们在cryo-EM分析之前应用此方法优化GPR119-G复合物样品，仅从在200 kV Glacios上收集的396个电影中获得了分辨率为3.51 Å的结构。这项研究表明，OBE-nMS+DMT方法成为在GPCRs和其他膜蛋白复合物的cryo-EM研究中预筛选样品条件的强大工具。
