In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes, that by assembling onto specialized Cenp-A nucleosomes 1,2 , function to connect centromeric chromatin to microtubules of the mitotic spindle 3,4 . Whereas the centromeres of vertebrate chromosomes comprise Mb of DNA and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5,6 . All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-A Nuc ), each of which is perfectly centred on its cognate centromere [7][8][9] . The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here, we describe the cryo-EM structure of the S. cerevisiae inner kinetochore module -the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-A Nuc ). The structure explains the inter-dependency of CCAN's constituent sub-complexes and shows how the 'Y'-shaped opening of CCAN accommodates Cenp-A Nuc to allow specific Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Crosslinking mass spectrometry (XL-MS) has received considerable interest due to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison to other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by use of bi-functional crosslinking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by mass spectrometry. XL-MS has received the required boost to tackle more complex samples with recent advances in crosslinking chemistry with MS-cleavable or reporter-based crosslinkers and faster, more sensitive and more versatile mass spectrometry platforms. This protocol provides a detailed description of our optimized conditions for a full proteome native protein preparation followed by crosslinking using the gas-phase cleavable crosslinking reagent DSSO. Following crosslinking, we demonstrate extensive sample fractionation and significantly simplified data analysis with XlinkX in Proteome Discoverer and subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within approximately 10 d including structural investigations.

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases -the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time.

Cells organize their actions partly through tightly controlled protein-protein interactionscollectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.