The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4–7 Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more “native” like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarises some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method. Micelles and classical detergent techniquesMembrane proteins account for~30% of both prokaryotic and eukaryotic proteins [1]. Integral proteins such as transporters or ion channels, as well as peripheral membrane proteins such as G-proteins, all perform essential tasks in signal transduction, cell metabolism and transport of small molecules [2][3][4]. Integral and peripheral membrane proteins are respectively embedded in or closely associated with the phospholipid bilayer of cell membranes. Therefore, their function often relies on their precise lipid environment [5,6]. For instance, cardiolipin, which constitutes about 20% of the inner mitochondrial membrane, is essential to the function of many mitochondrial transporters such as the ADP/ATP carriers, the enzymes of the respiratory chain, and bacterial proteins [7][8][9]. This is because cardiolipin offers polar and electrostatic interactions that increase protein stability[10]; similar interactions have been observed for other lipids [11,12]. Unfortunately, classical techniques to study membrane proteins involve the use of detergents that solubilise the protein but also destabilise its interaction with membrane lipids. In many cases, membrane proteins may be stable in only a few detergents, limiting the range of conditions that can be used for crystallization trials [13]. Consequently, much time is spent testing various detergents in different ratios and concentrations in the hope of finding conditions that will mimic the essential interactions of the protein with its natural lipidic environment -and this way stabilise the protein and preserve its functional state. These problems have hindered membrane protein research for many years: both biophysical characterisation and structure solution have suffered due to difficulties of extracting proteins from membranes and keeping them stable away from their native environment. The past few years have seen the emergence of new techniques aimed at providing a membrane-like natural environment. These novel techniques include liposomes, bicelles, discs, polymer and lipids based strategies.

Salmonella is an important genus of Gram-negative pathogens, treatment of which has become problematic due to increases in antimicrobial resistance. This is partly attributable to the overexpression of tripartite efflux pumps, particularly the constitutively expressed AcrAB-TolC. Despite its clinical importance, the structure of the Salmonella AcrB transporter remained unknown to-date, with much of our structural understanding coming from the Escherichia coli orthologue. Here, by taking advantage of the styrene maleic acid (SMA) technology to isolate membrane proteins with closely associated lipids, we report the very first experimental structure of Salmonella AcrB transporter. Furthermore, this novel structure provides additional insight into mechanisms of drug efflux as it bears the mutation (G288D), originating from a clinical isolate of Salmonella Typhimurium presenting an increased resistance to fluoroquinolones. Experimental data are complemented by state-of-the-art molecular dynamics (MD) simulations on both the wild type and G288D variant of Salmonella AcrB. Together, these reveal several important differences with respect to the E. coli protein, providing insights into the role of the G288D mutation in increasing drug efflux and extending our understanding of the mechanisms underlying antibiotic resistance.