Stabilization of multidrug efflux transporter MdfA and antibody fragments complex by cross-linking for Cryo-EM single-particle analysis.

Antibody fragments have proven to be highly valuable tools for the structural analysis of membrane proteins due to their distinctive ability to recognize and stabilize specific three-dimensional conformations. They can also act as crystallization chaperones in X-ray crystallography, aiding antibody-mediated crystal contacts. While recent advancements in cryogenic electron microscopy (cryo-EM) have revolutionized the analysis of membrane proteins, structure determination of small-sized membrane proteins remain a challenging task. Antibodies continue to be effective tools in this regard, especially for small membrane proteins. They increase overall particle sizes, assist in identifying transmembrane helices and the orientation of membrane proteins relative to the lipid bilayer, and help in particle image alignment. However, during sample preparation stages like purification and grid preparation, antibodies may dissociate from target membrane proteins, which can complicate high-resolution 3D structure determination. In this chapter, we will address these challenges by using Major Facilitator Superfamily (MFS) drug efflux transporter MdfA (∼44 kDa) complexed with Fab fragment as a case in point. We will provide a detailed protocol for stabilizing MdfA-Fab antibody fragment complexes using cross-linking reactions and following Cryo-EM single particle analysis. This method allows for efficient structure determination through cryo-EM single-particle analysis, making it useful for antibodies with weak binding capability. This procedure paves the way for advancing high-resolution structural investigations of small membrane proteins through cryo-EM single-particle analysis.