Automated structure modeling of large protein assemblies using crosslinks as distance restraints

Abstract

Crosslinking mass spectrometry is increasingly used for structural characterization of multisubunit protein complexes. Chemical crosslinking captures conformational heterogeneity, which typically results in conflicting crosslinks that cannot be satisfied in a single model, making detailed modeling a challenging task. Here we introduce an automated modeling method dedicated to large protein assemblies ('XL-MOD' software is available at http://aria.pasteur.fr/supplementary-data/x-links) that (i) uses a form of spatial restraints that realistically reflects the distribution of experimentally observed crosslinked distances; (ii) automatically deals with ambiguous and/or conflicting crosslinks and identifies alternative conformations within a Bayesian framework; and (iii) allows subunit structures to be flexible during conformational sampling. We demonstrate our method by testing it on known structures and available crosslinking data. We also crosslinked and modeled the 17-subunit yeast RNA polymerase III at atomic resolution; the resulting model agrees remarkably well with recently published cryoelectron microscopy structures and provides additional insights into the polymerase structure.

Modeling Protein Excited-state Structures from “Over-length” Chemical Cross-links

“…CXMS-based structural modeling has been explored using various software tools, yet a lot of these studies aimed for a single structure that had the lowest energy and best satisfied the experimental data (11,13,24,25). To fully account for the intramolecular cross-links, we developed the DynaXL method.…”

“…Recently it has been shown that for protein-protein complexes, intermolecular cross-links incompatible with a unique complex structure may arise from some alternative arrangement(s) of the complex (11,20). We thus reason that overlength intramolecular cross-links can be explained by the dynamic movement between different parts of the protein.…”

“…The cross-linked residues that are identified by high resolution mass spectroscopy should be close to each other, within the reach of the cross-linker used. However, it has been shown that the straight-line distance between the C␣ atoms of cross-linked residues calculated from the known structure sometimes exceed the maximum length of the cross-linker (10,11). The discrepancy between experimental cross-links and protein structures may be due to false identifications of the cross-links.…”

Bayesian Modeling of Biomolecular Assemblies with Cryo-EM Maps

“…A common scenario is to combine cryo-EM with crosslinking data, which also needs to be tested systematically with ISD. A Bayesian approach to modeling macromolecular assemblies with crosslinking data has been proposed recently by Ferber et al (2016). …”

Integrative modelling of cellular assemblies

“…The model was successfully validated with independent experimental data on protein–protein interactions. XL-MOD [53] also uses a Bayesian framework to reweight conflicting or ambiguous distance restraints from XL-MS, represented as log-harmonic potentials. An elastic network model based on a low-resolution representation of the subunit structures is used to sample conformational changes upon assembly and the component positions and orientations are sampled by a Monte-Carlo optimization scheme followed by relaxation using molecular dynamics.…”