Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.