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.
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Peisley, A., Skiniotis, G. (2015). 2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy. In: Filizola, M. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 1335. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2914-6_3
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DOI: https://doi.org/10.1007/978-1-4939-2914-6_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2913-9
Online ISBN: 978-1-4939-2914-6
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