title: 2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy.
abstract: While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (<150 kDa) complexes between GPCRs and their partner proteins. Negative stain EM, whereby the biological samples are embedded in a thin layer of heavy metal solution, is a well-established alternative technique that provides the enhanced contrast needed to visualize small macromolecular complexes. This methodology can offer a simple and powerful tool for the rapid evaluation of sample characteristics, such as homogeneity or oligomeric state. When coupled to single particle classification and averaging, negative stain EM can provide valuable information on the overall architecture and dynamics of protein complexes. Here we provide a concise protocol for negative stain imaging and two-dimensional (2D) projection analysis of GPCR complexes, including notes for the intricacies of the application in these biological systems.
authors.affiliation: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave, Ann Arbor, MI, 48109, USA.
authors.forename: Alys
authors.initials: A
authors.lastname: Peisley
authors.forename: Georgios
authors.initials: G
authors.lastname: Skiniotis
mesh_terms: D007091:Image Processing, Computer-Assisted / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013194:Staining and Labeling
chemical_list: D043562:Receptors, G-Protein-Coupled