Cryo-electron microscopy of membrane proteins

Abstract

Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.

Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

“…The larger volume was composed of mainly two connected domains, i.e., (i) a peripheral intense density originating from the GDN-detergent micellar headgroups and (ii) an inner, structured protein density ( Figure 4 A). This is common for detergent solubilized membrane protein cryo-EM volumes, since the hollow appearance between the micellar headgroup and the proteinaceous density is attributed to the hydrophobic portion of detergents [ 32 ]. Unlike in the structure of 4F2hc-LAT1 reported by Yan and colleagues [ 23 ], who also used GDN as a detergent for cryo-EM specimen preparation, the final volume of 4F2hc-LAT2 displayed a very strong micellar headgroup density ( Figure 4 A).…”

“…Although LAT1 and LAT2 are based on the primary amino acid sequence and are ≈48% identical and ≈65% similar to each other, the resolution of GDN purified 4F2hc-LAT2 was restricted to 7.5 Å, which is in stark contrast to published structures of GDN purified 4F2hc-LAT1 resolved at ≈3.3 Å [ 23 , 24 ]. It is known that in micelles, the scattering of electrons is particularly strong for the large hydrophilic headgroup [ 32 ], such as in GDN. Consequently, the usage of GDN is considered not to be an optimal detergent choice for the high-resolution structure solution of 4F2hc-LAT2.…”

The structural basis of T-cell receptor (TCR) activation: An enduring enigma

“…In that study, the authors determined an ensemble of cryoEM structures of yeast TRiC/ CCT at various nucleotide concentrations that included both open and closed states, revealing an unforeseen allosteric network at atomic resolution. Visualizing the cytoplasmic domains of CD3 subunits may require cryoEM analysis of the TCR-CD3 complex in nondetergent systems, such as nanodiscs or amphipols, that may more faithfully reproduce a natural lipid environment (67,71). Finally, none of the conventional structural methods used to study TCR triggering (X-ray crystallography, NMR, and cryoEM) take into account mechanical force, as posited in the mechanosensor model.…”

Reconstruction of destruction – in vitro reconstitution methods in autophagy research

“…Electron microscopy, electron tomography and subtomogram averaging are, in turn, powerful methods to reveal static high-resolution structures of proteins on membranes. Structural characterization of large membrane proteins by EM is achieved through nano-discs, which are lipid bilayer islands that are surrounded by amphipathic proteins, such as apolioprotein A1 (Thonghin et al, 2018). This suggests that the Atg1-Atg11 complex has two functions in autophagy: promoting autophagy initiation and regulating phagophore expansion.…”