The Blu-Ice and Distributed Control System (DCS) software packages were developed to provide unified control over the disparate hardware resources available at a macromolecular crystallography beamline. Blu-Ice is a user interface that provides scientific experimenters and beamline support staff with intuitive graphical tools for collecting diffraction data and configuring beamlines for experiments. Blu-Ice communicates with the hardware at a beamline via DCS, an instrument-control and data-acquisition package designed to integrate hardware resources in a highly heterogeneous networked computing environment. Together, Blu-Ice and DCS provide a flexible platform for increasing the ease of use, the level of automation and the remote accessibility of beamlines. Blu-Ice and DCS are currently installed on four Stanford Synchrotron Radiation Laboratory crystallographic beamlines and are being implemented at sister light sources.
Summary Synaptotagmin-1 and neuronal SNARE proteins play key roles in evoked synchronous neurotransmitter release. However, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains. The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms prior to Ca2+-triggering, and moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.
Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the Ϸ650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a -cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer. diabetes ͉ FeS cluster ͉ iron homeostasis ͉ thiazolidinedione ͉ oxidative stress I ron (Fe) is a vital trace element for virtually all organisms. Incorporation of this transition metal into iron-sulfur (Fe-S) clusters forms cofactors integral to diverse biological pathways involved in the capture and metabolism of light and chemical energy (1, 2). Because free iron can be highly toxic, an elaborate array of proteins has evolved to facilitate the transfer of iron through cell compartments, to insert iron into Fe-S clusters, and to incorporate Fe-S clusters into proteins. Fe-S cluster assembly takes place primarily, although not exclusively, within the mitochondrial matrix of eukaryotic cells, and defects in mitochondrial cluster assembly and export have profound consequences for rates of growth, iron accumulation, oxidative stress, and heme biosynthesis (1, 2).Mitochondrial dysfunction is associated with insulin resistance and the development of type 2 diabetes (3). Recent studies suggest that disease pathogenesis involves diminished mitochondrial oxidative capacity in insulin-sensitive tissues. Pharmacologic agents extensively used to treat insulin resistance such as the thiazolidinedione (TZD) pioglitazone are known to enhance oxidative capacity and normalize lipid metabolism (4, 5). Although TZDs are conventionally thought to operate through binding to peroxisome proliferator-activated receptors, a recent study by Colca and colleagues (6) identified an additional binding target within mitochondrial membranes that was named mitoNEET, on the basis of the subcellular localization (mito) and the presence of the amino acid sequence Asn-Glu-Glu-Thr (NEET).MitoNEET was determined to be an integral protein of the outer mitochondrial membrane (OMM) by a series of studies, including immuno-electron microscopy and detailed fractionation studies of highly purified rat liver mitochondria. An amino-terminal signal sequence within the first 32 residues, containing a predicted transmembrane domain, targets mitoNEET to the outer membrane. The orientation of this protein toward the cytoplasm was established by proteolytic digestion...
An automated system for mounting and dismounting pre-frozen crystals has been implemented at the Stanford Synchrotron Radiation Laboratory (SSRL). It is based on a small industrial robot and compact cylindrical cassettes, each holding up to 96 crystals mounted on Hampton Research sample pins. For easy shipping and storage, the cassette ®ts inside several popular dry-shippers and long-term storage Dewars. A dispensing Dewar holds up to three cassettes in liquid nitrogen adjacent to the beamline goniometer. The robot uses a permanent magnet tool to extract samples from, and insert samples into a cassette, and a cryo-tong tool to transfer them to and from the beamline goniometer. The system is simple, with few moving parts, reliable in operation and convenient to use.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
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