## 5 Membrane Proteins in SMALPs  
At least 40 different membrane proteins expressed in bacterial, yeast, insect, and mammalian cells have been solubilized using SMA for detailed biochemical analysis. Entire lipid rafts can be solubilized intact using SMA with no detergent exposure, allowing the protein:lipid bilayer patches to be examined. The solubilization of T cell membranes by addition of SMA(3:1) at 1% (\(w/v\)) yields >250 nm sized fragments that can be immunoprecipitated to isolate those with Src family kinases and glycosylphosphatidylinositol-anchored proteins [44]. These \"raft\" nanodiscs are the largest observed to date and contain ordered lipids including an excess of cholesterol, phosphatidylerines, sphingomyelins, ceramides, and monohexosylceramides compared to \(\sim\)20 nm nanodiscs, which contain relatively more phosphatidylcholine, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) lipids. Thus, SMA provides a detergent-free approach to solubilize both large lipid rafts and smaller nanodomains with distinct groups of lipids and compare those associated with specific transmembrane proteins. Structures of diverse memteins in SMALPs have been solved using various high-resolution methods, indicating the potential feasibility of the SMALP approach for membrane proteomics. Several notable recent reports are discussed below to illustrate the potential of the technique and build on dozens of earlier studies of expressed and endogenous proteins solubilized in SMA for structure-function analysis. The preparation of the multidrug exporter AcrB in SMA2000 yielded a structure of the asymmetric trimer at 3.0 A resolution [45]. Its detergent-free purification from Escherichia coli membrane involved single-step metal affinity chromatography of the SMALPs which could then be directly applied to grids to collect cryo-EM data. Unlike detergent approaches, this reveals the intact memtein, which is 12 nm in diameter. It contains 31 visible lipids around the protein trimer, which is 9 nm in diameter The native lipid bilayer is highly organized with 24 lipid molecules engaging protein residues and being packed into hexagonal arrays within the inner leaflet and an ordered irregular array in the outer leaflet (Figure 3). Although the polymers and lipid headgroup identities are not discernible, the development of more homogeneous polymers is likely to improve resolution further. The largest SMALP-based structure is that of the Alternative Complex III (ACIII) supercomplex, which was resolved at 3.4 A resolution using cryoEM [17]. This study used SMA3000 as well as XIRAN 25010 to prepare functional complexes of this 464 kDa memtein and revealed the positions of acylated residues mediating photosynthetic electron transport as well as multiple phospholipids haem groups and iron-sulfur clusters. The SMALP contains this entire 9 \(\times\) 13 nm assembly with its 6 protein subunits, 48 transmembrane helices, and accessory cytochrome c oxidase within a shell of native lipid that were extracted together from the membrane. This demonstrates the adaptability of the SMA nanoparticles, which is shown to conform to the irregular dimensions of membrane protein multimers. The G protein-coupled receptor (GPCR) superfamily includes many valuable targets for drug discovery. The first 3D structure of a seven transmembrane helix protein that had been extracted with SMA(3:1) was elucidated using XRD at 2.0 A resolution, which is better than that of detergent-extracted protein in crystals [28]. The seven transmembrane helices of this microbial rhodopsin protein are arranged in a trimer that binds trans-retinal as well as monoolein molecules which were used to prepare the lipidic cubic phase for in meso crystallization. Bound bacterial lipids could not be resolved, presumably due to their displacement by the excess monoolein molecules. A heterotetrameric GPCR complex that recognizes the growth hormone secretagogue and dopamine can be solubilized using SMA (2:1) and binds to G proteins [46].