Membrane protein structure and dynamics probed by MicroED

Membrane proteins are essential to cellular signaling, transport, and homeostasis, yet their amphipathic nature, dependence on lipids or detergents, and typically difficult expression and purification make them difficult targets for structural methods such as X-ray crystallography. Moreover, most membrane proteins in the human proteome are too small for investigation by single-particle electron cryomicroscopy methods. Microcrystal Electron Diffraction (MicroED) has emerged as a powerful method for overcoming these barriers, allowing structure determination from nanocrystals of membrane proteins embedded in the near-native environment of the lipid membrane. In the present review, we discuss how recent improvements in MicroED, such as focused ion-beam milling and high-throughput data collection approaches, facilitate structure determination and investigation of protein dynamics. We focus on applications involving junction-forming proteins, G protein-coupled receptors, and ion channels, where MicroED has revealed physiologically relevant assemblies, lipid interactions, and transient functional states that were not attainable by other structural biology methods.