Cryo-electron microscopy (Cryo-EM) structural determination of the MmpL family of transporters

Single-particle cryo-electron microscopy (cryo-EM) has become a very powerful technique in the field of membrane protein structural biology. Historically, protein structure determination requires homogenous and pure samples, and sample heterogeneity often hampered the progress of drug design and development, especially those targeting membrane proteins and their complexes. With the rapid development of the instrumentation, software and methodologies of cryo-EM, it is now feasible to obtain high-resolution cryo-EM structural information of membrane proteins, from both pure/homogeneous and impure/heterogeneous samples. Here, we present our current protocols and methodologies for this structural technique. Case studies show step-by-step how we used this cryo-EM methodology to elucidate the structure and assembly of the important mycobacterial membrane protein large (MmpL) family of transporters. It is our intention to encourage more researchers to employ a variety of structural, biophysical and biochemical methodologies to continue to study critical membrane proteins for the development of novel therapeutic strategies to combat human diseases.