X-ray crystallography and electron microscopy maps resolved to 3-8 Å are generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

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.