Final Activity Report Summary - CRYO-EM URI-COMPLEX (A Combined Cryo Electron Microscopy and Mass Spectrometry Approach for the Structural and Functional Characterization of the URI-complex)
We proposed an integrated approach for the structure determination of protein complexes that combines the strength of two technologies. While mass spectrometry can identify and quantify proteins and protein interactions with high sensitivity in the bulk of lysate of many cells, cryo electron microscopy can in principle detect individual protein complexes. We chose of the human pathogen Leptospira interrogans as a model system and determined the cytoplasmic protein concentrations of several protein complexes using selected reaction monitoring mass spectrometry.
This provided information aided the identification of individual protein complexes within cryo electron tomograms of Leptospira interrogans cells by template matching. Furthermore, we identified the interfaces within several protein complexes, including the URI complex, using isotope coded cross-linking reagents with sub-sequent identification of cross-linked peptides by tandem mass spectrometry. The outlined technology promises to establish the critical link from structural analysis of isolated protein complexes to their observation in the cell.
This provided information aided the identification of individual protein complexes within cryo electron tomograms of Leptospira interrogans cells by template matching. Furthermore, we identified the interfaces within several protein complexes, including the URI complex, using isotope coded cross-linking reagents with sub-sequent identification of cross-linked peptides by tandem mass spectrometry. The outlined technology promises to establish the critical link from structural analysis of isolated protein complexes to their observation in the cell.