Acta crystallographica. Section D, Biological crystallography最新文献

High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

A novel approach is presented for an a priori assessment of the ambiguity associated with spherically averaged single-particle scattering. The approach is of broad interest to the structural biology community, allowing the rapid and model-independent assessment of the inherent non-uniqueness of three-dimensional shape reconstruction from scattering experiments on solutions of biological macromolecules. One-dimensional scattering curves recorded from monodisperse systems are nowadays routinely utilized to generate low-resolution particle shapes, but the potential ambiguity of such reconstructions remains a major issue. At present, the (non)uniqueness can only be assessed by a posteriori comparison and averaging of repetitive Monte Carlo-based shape-determination runs. The new a priori ambiguity measure is based on the number of distinct shape categories compatible with a given data set. For this purpose, a comprehensive library of over 14,000 shape topologies has been generated containing up to seven beads closely packed on a hexagonal grid. The computed scattering curves rescaled to keep only the shape topology rather than the overall size information provide a `scattering map' of this set of shapes. For a given scattering data set, one rapidly obtains the number of neighbours in the map and the associated shape topologies such that in addition to providing a quantitative ambiguity measure the algorithm may also serve as an alternative shape-analysis tool. The approach has been validated in model calculations on geometrical bodies and its usefulness is further demonstrated on a number of experimental X-ray scattering data sets from proteins in solution. A quantitative ambiguity score (a-score) is introduced to provide immediate and convenient guidance to the user on the uniqueness of the ab initio shape reconstruction from the given data set.

High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort. REdiii is a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies in case the preconfigured solution is inadequate. This integrated pipeline is targeted at providing a comprehensive and efficient approach to screening for ligand-bound co-crystal structures while minimizing repetitiveness and allowing a high-throughput scientific discovery process.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery.

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.

Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalous diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.

Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

Carp FEL (fishelectin or fish-egg lectin) is a 238-amino-acid lectin that can be purified from fish eggs by exploiting its selective binding to Sepharose followed by elution with N-acetylglucosamine. Its amino-acid sequence and other biochemical properties have previously been reported. The glycoprotein has four disulfide bridges and the structure of the oligosaccharides linked to Asn27 has been described. Here, the three-dimensional structures of apo carp FEL (cFEL) and of its complex with N-acetylglucosamine determined by X-ray crystallography at resolutions of 1.35 and 1.70 Å, respectively, are reported. The molecule folds as a six-bladed β-propeller and internal short consensus amino-acid sequences have been identified in all of the blades. A calcium atom binds at the bottom of the funnel-shaped tunnel located in the centre of the propeller. Two ligand-binding sites, α and β, are present in each of the two protomers in the dimer. The first site, α, is closer to the N-terminus of the chain and is located in the crevice between the second and the third blades, while the second site, β, is located between the fourth and the fifth blades. The amino acids that participate in the contacts have been identified, as well as the conserved water molecules in all of the sites. Both sites can bind the two anomers, α and β, of N-acetylglucosamine, as is clearly recognizable in the electron-density maps. The lectin presents sequence homology to members of the tachylectin family, which are known to have a function in the innate immune system of arthropods, and homologous genes are present in the genomes of other fish and amphibians. This structure is the first of a protein of this group and, given the degree of homology with other members of the family, it is expected that it will be useful to experimentally determine other crystal structures using the coordinates of cFEL as a search probe in molecular replacement.

Homoserine dehydrogenase (HSD) is an oxidoreductase in the aspartic acid pathway. This enzyme coordinates a critical branch point of the metabolic pathway that leads to the synthesis of bacterial cell-wall components such as L-lysine and m-DAP in addition to other amino acids such as L-threonine, L-methionine and L-isoleucine. Here, a structural rationale for the hydride-transfer step in the reaction mechanism of HSD is reported. The structure of Staphylococcus aureus HSD was determined at different pH conditions to understand the basis for the enhanced enzymatic activity at basic pH. An analysis of the crystal structure revealed that Lys105, which is located at the interface of the catalytic and cofactor-binding sites, could mediate the hydride-transfer step of the reaction mechanism. The role of Lys105 was subsequently confirmed by mutational analysis. Put together, these studies reveal the role of conserved water molecules and a lysine residue in hydride transfer between the substrate and the cofactor.