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.

Abstract. Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS 2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments.

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.

Setting Shambhala is a 5-day electronic dance music (EDM) festival held in rural British Columbia that annually hosts between 15,000 and 18,000 people on a 500-acre ranch. The AIDS Network Outreach & Support Society (ANKORS) has provided harm reduction services throughout the duration of the festival since 2003, including point-of-care drug checking, which allows realtime testing of illicit substances to assess their composition. Drug checking results are provided directly to clients and displayed in aggregate on a screen for all attendees to see. Intervention In 2017, ANKORS added fentanyl checking to their repertoire of drug checking technologies for festivalgoers. Volunteers used a brief survey to collect information on what clients expected the samples to contain. Volunteers carried out drug checks and subsequently logged test results. ANKORS provided an amnesty bin at the tent for clients who chose to discard their substances. Outcomes Of the 2683 surveys, 2387 included data on both the client's belief and the actual test result. Clients were more likely to discard when the test result differed from their belief (5.16%) than when their belief was confirmed (0.69%). Discarding increased to 15.54% when the test could not clearly identify a substance and to 30.77% if the client did not have a prior belief of the substance. Of 1971 samples tested for fentanyl, 31 tested positive and 16.13% of clients discarded compared to 2.63% in the negative group. Implications Drug checking services appeal to festivalgoers who, when faced with uncertainty, may discard their substances. This innovative harm reduction service allows for a personalized risk discussion, potentially reaching others via word-of-mouth and early warning systems. Résumé Contexte Shambhala est un festival de musique électronique qui se déroule sur cinq jours dans un ranch de 500 acres en campagne britanno-colombienne et qui accueille de 15 000 à 18 000 personnes. L'AIDS Network Outreach & Support Society (ANKORS) offre des services de réduction des méfaits pour toute la durée du festival depuis 2003, notamment sous forme d'analyses en temps réel de la composition de substances illicites dans des points de service. Les résultats sont remis directement aux clients et affichés sous leur forme brute dans un tableau général que peuvent voir tous les festivaliers.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.