Protein complexes in cells by AI-assisted structural proteomics

Francis J. O'Reilly, Andrea Graziadei, Christian Forbrig, Rica Bremenkamp, Kristine Charles, Swantje Lenz, Christoph Elfmann, Lutz Fischer, Jörg Stülke, Juri Rappsilber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry and co-fractionation mass spectrometry (CoFrac-MS) to identify protein–protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein–protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Original languageEnglish
Article numbere11544
Pages (from-to)e11544
Number of pages20
JournalMolecular Systems Biology
Issue number4
Publication statusPublished - 12 Apr 2023

Keywords / Materials (for Non-textual outputs)

  • alphaFold-multimer
  • crosslinking mass spectrometry
  • protein–protein interactions
  • pyruvate dehydrogenase
  • uncharacterized proteins


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