Highlights d One copy of CENP-N is lost for every two copies of CENP-C at mitotic onset d Structures of core centromeric nucleosome complex (CCNC) with 1 or 2 copies CENP-N d Natural centromere nucleosomal DNA conformation corresponds to robust CCNC assembly d Emerging model of mitotic centromeric chromatin with global and local asymmetries

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein–protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication–transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.