Abstract
The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, 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 disuccinimidyl diacetic 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.
Highlights
Ribosomes, the molecular machines that are responsible for protein synthesis, have frequently attracted interest both from a biological[1] as well as from a methodological perspective[2,3,4]
We highlight the broad synergy of XL-MS with high-resolution structural methods, as our XL-MS experiments allowed remodeling of 2,057 residues in total, optimizing the current working model of the E. coli ribosome (Workflow, Fig. 1)
We first analyzed the intramolecular cross-links. 126 non-redundant intramolecular cross-links were mapped onto 142 ribosome structures, while only 11% (14/126) of these cross-links could not be mapped to any available structure (Table S2)
Summary
The molecular machines that are responsible for protein synthesis, have frequently attracted interest both from a biological[1] as well as from a methodological perspective[2,3,4]. The usefulness of FDR calculations and their correlation to true positive protein–protein interactions and the corresponding structural models[29] as well as the choice of molecular models that are used for cross-link mapping are in some cases suboptimal This is because (a) current studies have a bias for high-abundant proteins, but methods to address this issue are being implemented[30] and (b) few molecular models deposited in structure databases are being evaluated for cross-linking distances, and only one molecular model is selected for distance calculation per protein complex, despite the wealth of structural data. We highlight the broad synergy of XL-MS with high-resolution structural methods, as our XL-MS experiments allowed remodeling of 2,057 residues in total, optimizing the current working model of the E. coli ribosome (Workflow, Fig. 1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
