Capturing the native structure of membrane proteins

• 成果类型: Article
• 署名作者: Liu, Hang; Tse, Chun Mong; Dang, Shangyu
• 署名单位: Hong Kong University of Science & Technology; Hong Kong University of Science & Technology
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11436
• DOI: 10.1073/pnas.2423407122
• 发表日期: 2025-09-09
• 关键词: cryo-em structure; Visualization; lysozyme; crystal; SYSTEM; reconstruction; purification; tomography; detergents; CHALLENGES

摘要

Membrane proteins play crucial roles in numerous biological processes and are important drug targets. However, structural studies of membrane proteins often rely on solubilization with detergents, which may not accurately reflect their native states in a cellular context. Additionally, identifying suitable detergents for individual membrane proteins can be a detailed and time-consuming process. Here, we developed a vesicle-based method that preserves the native lipid environment for subsequent structural and functional studies. Using the bacterial multidrug efflux transporter AcrB as an example, we isolated AcrB-containing vesicles and determined its cryo-EM structure with all protomers in a loose (L) state at 3.88 & Aring; by incorporating our micrograph-based sorting strategy. Notably, compared to the L-state AcrB in liposomes and nanoparticles, the exterior transmembrane helices (TMs) in our map exhibited superior quality, featuring a continuous and clear representation of l alpha, which is positioned horizontally within the lipid bilayer. We further expanded our method by identifying endogenous membrane proteins, including F-ATPase and respiratory complexes, in vesicles generated using mitochondria from pig hearts. The high-resolution structure of respiratory complex III in vesicles revealed a shared subunit nine between two monomers. Briefly, our method presents a promising and straightforward approach for studying the structure and function of membrane proteins in their native environment, eliminating the need for detergent screening and protein purification.