Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

Abstract

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 biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…Sedimentation velocity experiment reveals the presence of dimeric and trimeric forms, while negative stain EM and 3D reconstruction reveals an inner vestibule surrounded by a belt of at least 40 lipid molecules and polymer around the trimeric protein (Figure 4b). The global structure of AcrB extracted, purified and visualized in SMA(2:1) is resolvable by cryo-EM at 8.8 Å, with higher resolution for its soluble domain while two exposed helices appear dynamic [31]. Together with recent progress on visualizing NMR signals of proteins in SMA-derived nanodiscs [22] and polymer developments, this bodes well for further structural studies of native membrane states.…”

Examining the stability of membrane proteins within SMALPs

“…To date SMA solubilisation and subsequent affinity purification has been demonstrated to be effective for a wide range of proteins including transporters, ion channels, enzymes and G-protein coupled receptors (GPCRs), from many different expression systems [6][7][8][9][10][11][12]. SMALPs are small and soluble particles which are amenable to many downstream techniques including spectroscopy and ligand binding assays [6,8,9,11,13], as well as structural studies both by crystallography and electron microscopy [6,12,[14][15][16]. SMALPencapsulated proteins have been shown to have an enhanced stability compared to traditional detergent approaches [6][7][8][9]17].…”

Structure of the alternative complex III in a supercomplex with cytochrome oxidase

“…Although ACIII has eluded structural characterization, several of its subunits are homologous to members of the CISM (Complex Iron-Sulfur Molybdoenzyme) superfamily 8 , including the proton pump polysulfide reductase 9,10 . We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid (SMA) copolymer 11–14 , both as an independent enzyme and as a functional 1:1 supercomplex with an aa 3 -type cytochrome c oxidase (cyt aa 3 ). We determined the structure of ACIII to 3.4 Å resolution by cryo-EM and constructed an atomic model for its six subunits.…”