Combining cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (CX-MS) for structural elucidation of large protein assemblies

Determining the structures of, and gaining insight into, the function of large protein complexes at the molecular or atomic level has become a key part of modern structural biology. Electron cryo-microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes that are not feasible with other structural techniques like X-ray of crystallized proteins (protein complexes) or nuclear magnetic resonance (NMR) spectroscopy of proteins (protein complexes) in solution. To resolve the regions that are less well defined in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the proximity between amino-acid residues as distance constraints for homology or de novo modelling. The CX-MS strategy involves covalent linkage, with chemical cross-linkers, of residues close to each other in three-dimensional space and identifying these connections by mass spectrometry. In this article, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction. We further evaluate a number of important examples of structure determination that followed this combinatorial strategy.