Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544
Proc Natl Acad Sci U S A. 2020 Aug 4;117(31):18497-18503. doi: 10.1073/pnas.2009385117. Epub 2020 Jul 17.
Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
膜蛋白（MPs）曾经是 X 射线晶体学为主流方法时结构生物学中最难的靶标。随着单颗粒电子冷冻电镜（cryo-EM）分辨率的革命，孤立 MPs 的结构解析取得了快速进展。下一个挑战是保留电化学梯度和膜曲率，以便对依赖这些化学和物理性质发挥其生物学功能的 MPs 进行全面的结构解析。为此，我们提出了一种在脂质体中嵌入 MPs 的 cryo-EM 结构分析的便捷工作流程，以具有良好特征的 AcrB 作为原型。通过优化的脂蛋白体分离、在石墨烯网格上的 cryo 样品制备以及高效的颗粒选择策略，获得了在 3.9 Å 分辨率下嵌入脂质体的 AcrB 的三维（3D）重建。当周围膜显示不同曲率时，三聚体 AcrB 的构象保持不变。我们的方法可以广泛应用于具有独特可溶性结构域的 MPs 的 cryo-EM 分析，为 cryo-EM 分析受跨膜电化学梯度和/或膜曲率影响的整合或周边 MPs 的功能奠定了基础。
