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bioRxiv · 10.1101/2020.05.04.077503

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Tueting, C.·Iacobucci, C.·Ihling, C. H.·Kastritis, P. L.·Sinz, A.
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Abstract

The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

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Tueting, C., Iacobucci, C., Ihling, C. H., Kastritis, P. L., Sinz, A.. 2020-05-05. Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity. https://doi.org/10.1101/2020.05.04.077503

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