The crystal structure of a rice CesA8 plant-conserved region (P-CR), solved to 2.4 Å resolution, is dockedto a model of the catalytic domain of CesA8 using SAXS molecular envelopes.
The ...crystallographic structure of a rice (
Oryza sativa
) cellulose synthase, OsCesA8, plant-conserved region (
P-CR
), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The
P-CR
structure was fit into a molecular envelope for the
P-CR
domain derived from small-angle X-ray scattering data. The
P-CR
structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the
P-CR
domain from the molecular docking models places the
P-CR
connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the
P-CR
coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.
Crystallographic fragment screening is a technique for initiating drug discovery in which protein crystals are soaked or grown with high concentrations of small molecule compounds (typically MW ...110-250 Da) chosen to represent fragments of potential drugs. Specific binding of these compounds to the protein is subsequently visualized in electron density maps obtained from analysis of X-ray diffraction data collected from these crystals. Theoretical and practical experience indicate that a suitably diverse library of fragment compounds containing only a few hundred compounds may be sufficient to provide a comprehensive screen of the protein target. By soaking crystals in mixtures of 3-10 compounds a fragment screen may be completed within ∼100 diffraction data sets. This data collection requirement may be met given reproducible well-diffracting protein crystals and robotic sample handling equipment at a high flux X-ray source. The leading practical issue for most crystallography laboratories that wish to launch a fragment screening project is the design and/or procurement of an appropriate fragment library. Although several off-the-shelf fragment libraries are available from chemical suppliers, the numbers, sizes, and solubility of the compounds in relatively few of these libraries are well-match to the specific needs of the crystallographic screening experiment. Informed consideration of the properties of compounds in the screening library, possibly augmented by additional filtering of available compounds with appropriate search tools, is required to design a successful experiment. The analysis of results from crystallographic fragment screening involves highly repetitive application of routine image data processing and structure refinement calculations from many very similar crystals. Efficient handling of the data applies a high-throughput structure determination methodology that conveniently packages the structure solution calculations into a single process that provides the crystallographer-analyst with ready-to-view maps for evaluating crystals for bound compounds.
Mineral inclusions in biomass are attracting increased scrutiny due to their potential impact on processing methods designed to provide renewable feedstocks for the production of chemicals and fuels. ...These inclusions are often sculpted by the plant into shapes required to support functional roles that include the storage of specific elements, strengthening of the plant structure, and providing a defense against pathogens and herbivores. In situ characterization of these inclusions faces substantial challenges since they are embedded in an opaque, complex polymeric matrix. Here we describe the use of Bragg coherent diffraction imaging (BCDI) to study mineral inclusions within intact maize stalks. Three-dimensional BCDI data sets were collected and used to reconstruct images of mineral inclusions at 50-100 nm resolution. Asymmetries in the intensity distributions around the Bragg peaks provided detailed information about the deformation fields within these crystal particles revealing lattice defects that result in distinct internal crystal domains.
Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are ...then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.
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