Wellcome Senior Research Fellow
Juri Rappsilber is a Wellcome Senior Research Fellow, Professor of Proteomics in the University of Edinburgh and Professor of Bioanalytics in the Technische Universität Berlin. His group develops tools to study the function, location, interactions and structure of proteins in cells. Most of the work involves cross-linking, mass spectrometry, machine learning and software development. Juri Rappsilber took his PhD in Proteomics at the European Molecular Biology Laboratory (EMBL) in Heidelberg and Goethe Universität Frankfurt am Main in the lab of Matthias Mann and followed him as a postdoc to Odense, Denmark. In 2003 he relocated as principle investigator to the FIRC Institute of Molecular Oncology, Milan, which he left in 2006 to move to the University of Edinburgh. He became Wellcome Senior Research Fellow in 2009 and Professor of Proteomics in 2010. He has also held the post of Professor of Bioanalytics in Berlin since 2011.
Structural biology has made amazing advances on providing us models of proteins alone or in complexes by technologies such as crystallography, electron microscopy or nuclear magnetic resonance spectroscopy. These methods continue to advance yet fail mostly on requiring proteins to be extracted from their native environments, a process that many proteins do not honour with keeping their native structure. We are one of the pioneers of developing crosslinking mass spectrometry as an alternative approach. This sees proteins crosslinked in their native environment and the sites of crosslinking then being determined by mass spectrometry and data analysis. We are optimising many steps of this process: choice of crosslink reagents, protein-to-reagent ratio, digestion conditions, sample preparation, mass spectrometric acquisition, peak picking, data base construction. We are also further developing our search software xiSEARCH and integrated data visualisation tool xiVIEW. We fully embrace ideas of open sharing our insights and tools by making use of preprint repositories, providing our code via GitHub, submitting our data to public repositories and establishing field standards in close collaboration with key stakeholders such as HUPO PSI and EBI. The continuous progress on our tools has allowed us now to move into (simple) cells.
Structural biology performed inside cells can capture molecular machines in action within their native context. We developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography (in collaboration with Julia Mahamid at the EMBL Heidelberg), and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell. This is in stalk contrast to structures obtained by single particle cryoEM using reconstituted transcription-translation in E. coli, which see NusG and not NusA at the nexus of RNAP and ribosome. Future work will have to clarify whether this difference can be accounted for by working with different species or that working in cells captures a different state then working in a reconstituted system.
Dau, T., Bartolomucci, G., Rappsilber, J. (2020) Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin. Anal Chem.92, 9523-9527.
O'Reilly, F.J., Xue, L., Graziadei, A., Sinn, L., Lenz, S., Tegunov, D., Blötz, C., Singh, N., Hagen, W.J.H., Cramer, P., Stülke, J., Mahamid, J., Rappsilber, J. (2020) In-cell architecture of an actively transcribing-translating expressome. Science.369, 554-557.
Leitner, A., Bonvin, A.M.J.J., Borchers, C.H., Chalkley, R.J., Chamot-Rooke, J., Combe, C.W., Cox, J., Dong, M.Q., Fischer, L., Götze, M., Gozzo, F.C., Heck, A.J.R., Hoopmann, M.R., Huang, L., Ishihama, Y., Jones, A.R., Kalisman, N., Kohlbacher, O., Mechtler, K., Moritz, R.L., Netz, E., Novak, P., Petrotchenko, E., Sali, A., Scheltema, R.A., Schmidt, C., Schriemer, D., Sinz, A., Sobott, F., Stengel, F., Thalassinos, K., Urlaub, H., Viner, R., Vizcaíno, J.A., Wilkins, M.R., Rappsilber, J. (2020) Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry. Structure. 28, 1259-1268.
In-cell integrative structural biology reveals the structural basis of coupling between transcription and translation in M. pneumoniae.
A. 577 distinct PPIs identified at 5% PPI-level FDR (interactions to 8 abundant glycolytic enzymes and chaperones are removed for clarity). Membrane-associated proteins are shown in grey. Circle diameter indicates relative protein size. Blue: 50S ribosomal proteins; yellow: 30S ribosomal proteins; green: RNAP; orange: NusA. Each edge represents one or more crosslinks.
B. Interactors of RNAP and NusA. NusA NTD, S1, KH domains, and proline rich region (PR) are annotated. Line thickness represents the number of identified crosslinks.
C. Model of a RNAP-ribosome supercomplexe in untreated cells, the expressome compromises an actively elongating RNAP and ribosome.