Chapter title |
2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy
|
---|---|
Chapter number | 3 |
Book title |
G Protein-Coupled Receptors in Drug Discovery
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2914-6_3 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2913-9, 978-1-4939-2914-6
|
Authors |
Alys Peisley, Georgios Skiniotis, Peisley, Alys, Skiniotis, Georgios |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
United States | 1 | 4% |
Unknown | 27 | 96% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 8 | 29% |
Researcher | 5 | 18% |
Student > Bachelor | 4 | 14% |
Student > Doctoral Student | 2 | 7% |
Student > Postgraduate | 2 | 7% |
Other | 3 | 11% |
Unknown | 4 | 14% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 13 | 46% |
Agricultural and Biological Sciences | 6 | 21% |
Chemistry | 3 | 11% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 4% |
Unknown | 5 | 18% |