Processing Of X-ray Diffraction Data Research Articles

Possibilities of the X-ray diffraction data processing method for detecting reflections with intensity below the background noise component

The values of the signal-to-noise ratio are determined, at which the method of processing X-ray diffraction data reveals reflections with intensity less than the noise component of the background. The possibilities of the method are demonstrated on weak reflections of α-quartz. The method of processing X-ray diffraction data makes it possible to increase the possibilities of X-ray phase analysis in determining the qualitative phase composition of multiphase materials with a small (down to 0.1 wt.%) content of several (up to eight) phases.

Enhancing the Microhardness of Coatings Produced by Cold Gas Dynamic Spraying through Multi-Reinforcement with Aluminum Powders Containing Fullerenes and Aluminum Nitride

Coatings with high hardness were successfully obtained using low-pressure cold spray (LPCS) technology from nanocrystalline powders based on the aluminum alloy AlMg6, which were multi-reinforced with 0.3 wt.% fullerenes and 10–50 wt.% AlN. The powders were synthesized through a two-stage high-energy ball milling process, resulting in a complex mechanical mixture consisting of agglomerates and micro-sized ceramic particles of AlN. The agglomerates comprise particles of the nanocomposite material AlMg6/C60 with embedded and surface-located, micro-sized ceramic particles of AlN. Scanning electron microscopy and EDS analyses demonstrated a uniform distribution of reinforcing particles throughout the coating volume. An X-ray diffraction (XRD) analysis of the coatings revealed a change in the predominant orientation of matrix alloy grains to a more chaotic state during deformation over the course of cold gas dynamic spraying. A quantitative determination of AlN content in the coating was achieved through the processing of XRD data using the reference intensity ratio (RIR) method. It was found that the proportion of transferred ceramic particles from the multi-reinforced powder to the coating did not exceed ~65%. Experimental evidence indicated that LPCS processing of mono-reinforced nanocrystalline powder composite AlMg6/C60 practically did not lead to the formation of a coating on the substrate and was limited to a monolayer with a thickness of ~10 µm. The microhardness of the monolayer coating obtained from the deposition of AlMg6/C60 powder was 181 ± 12 HV. Additionally, the introduction of 10 to 50 wt.% AlN into the powder mixture contributed to the enhancement of growth efficiency and an increase in coating microhardness by ~1.4–1.7 times. The obtained results demonstrate that the utilization of agglomerated multi-reinforced powders for cold gas dynamic spraying can be an effective strategy for producing coatings and bulk materials based on aluminum and its alloys with high microhardness.

Synthesis and characterization of nano crystallite carboxamide-based iron(III) complexes: SOD mimetic activity, antibacterial and anticancer activity and molecular docking study

Three carboxamide-based ligands and their iron(III) complexes were prepared and structurally characterized. Analytical, thermal and mass spectra measurements showed a 1:1 stoichiometric (M:L) of the synthesized iron(III) complexes. The distorted octahedral geometry of the present iron(III) complexes was assigned based on the results of spectroscopy and magnetometry. Processing of X-ray diffraction data for powder samples by the software Expo 2014 confirmed the octahedral geometry of the three iron(III) complexes. Electrochemical properties of the present iron(III) complexes were studied by cyclic voltammetric measurements. The present iron(III) complexes exhibit SOD like activity with IC50 values of 16.45, 15.24 and 9.70 μM. The drive forces (−λ or ΔG°) controlling these biocatalytic reactions were determined and correlated with catalytic activity. The proposed catalytic mechanistic implications for the conversion of O2 •− to H2O2 and H2O were discussed. The antimicrobial activity has been studied in vitro against G(+) and G(−) pathogenic bacteria. The in vitro anticancer activity of the carboxamide-based ligands and their iron(III) complexes against human Hepatocellular carcinoma (HepG-2) cell lines was examined. The obtained results demonstrated the potent anticancer activity of iron(III) complexes with increased safety on normal cells compared to cisplatin. Molecular docking calculations confirmed the experimental findings of the antibacterial and anticancer activities of both free ligands and their iron(III) chelates. Communicated by Ramaswamy H. Sarma

Synthesis and characterization of naphthaldiimine-based ruthenium(III) complexes; homogenous catalytic hydrogenation and isomerization of internal and terminal alkenes

This study is concerned with synthesis and characterization of four naphthaldiimine ligands and their ruthenium(III) complexes. The tetradentate diimines were synthesized by a Schiff condensation reaction between 2-hydroxy-1-naphthaldehyde and various aliphatic diamines with chains of 2, 4, 5 and 6 carbon atoms. Analytical, mass spectra, thermal and electrolytic solution conductance measurements demonstrated the molecular formulas of the prepared Ru(III) complexes. A distorted octahedron stereochemistry was proposed for these six-coordinate naphthaldiimine-based Ru(III) complexes. Processing of X-ray diffraction data of 1 and 2 by the computer program Expo 2014 confirmed the final structural formulas that were determined based on spectroscopic and magnetic studies. The catalytic potential of the newly synthesized naphthaldiimine-based Ru(III) complexes in the hydrogenation of 1-hexene and cyclohexene was investigated. The Ru(III) catalysts catalyzed the double bond migration of 1-hexene to give trans-2-hexene which catalytically isomerizes to the stereo isomer cis-2-hexene. The induction step of the catalytic cycle is the formation of the actual catalyst, metal hydride (Ru(III)-H), that was generated in situ during the hydrogenation reaction. Several parameters controlling the catalytic reactions including the structure of the Ru(III) complex, the phase type of catalysis, and the quantity and nature of the solvents used were investigated. Various experiments made it possible to suggest a mechanism for the hydrogenation reactions.

Top-of-holes sensing techniques: developments within Deep Exploration Technologies Cooperative Research Centre

In this paper, we summarise advancements in top-of-hole sensing achieved within the Deep Exploration Technologies Cooperative Research Centre (DET CRC). It was demonstrated that the drill fines, which were previously discarded, show high potential to act as a representative sample media of the lithologies intersected by the drill hole and can be successfully used for analysis in real time. The Lab-at-Rig® (LAR®) system was developed for prospecting rigs (diamond drilling in the first instance and coil tube drilling in the future) and encompasses sample capture, sample preparation and presentation to sensors. In the initial setup of the LAR platform, there are two sensors, a portable X-ray fluorescence (pXRF) and a portable X-ray diffraction, capable of delivering chemical and mineralogical data in near real time. Laser-induced breakdown spectroscopy was also explored as a potential additional sensor for future versions of the LAR system, as it can yield information on elemental composition including essential light elements not currently measured by air-based pXRF detectors (e.g. Li, Na and Mg at low levels) or other elements problematic by pXRF (e.g. Au). The LAR system implements X-ray diffraction (XRD) analysis from which mineralogical data (mineral identification and most importantly mineral quantification) must be obtained in near real time. The existing challenge with XRD is that any data processing and especially data interpretation with available software packages requires some expertise in the field and background in crystallography and is time-consuming. Hence, SwiftMin®, the world’s first algorithm for automated processing of XRD data, was developed. It provides mineral identification and quantification and performs all calculations and processing independent from a user. SwiftMin returns a result in seconds and is able to batch process hundreds of XRD patterns in a matter of minutes. The above means, that SwiftMin is a technology that allows processing of large amount of XRD data quickly, saving time, costs and labour. The overall concept and vision developed within the DET CRC in top-of-hole sensing by coupling chemical and mineralogical analyses of drilling materials is to provide an end-to-end solution that supports rapid decision making by a geologist, at the time-scale of drilling the hole. KEY POINTS Lab-at-Rig® workflow results in geochemical and mineralogical analyses by the time the drill hole is completed, providing objective logging and an opportunity to make important decisions during the course of a drilling campaign. SwiftMin® capability to process the data quickly and with no user interaction will allow X-ray diffraction to become a routine and cost-effective technique for analysis of geological materials. Finally, while laser-induced breakdown spectroscopy results look promising, particularly for such elements as Na, Mg and Au, application of laser-induced breakdown spectroscopy for rapid, on-site analysis of geological materials requires some further research, above all on how to minimise the ‘matrix effects’.

Method of X-Ray Diffraction Data Processing for Multiphase Materials with Low Phase Contents

Amorphous, glass, and glass-ceramic materials practically always include a significant number (more than eight) of crystalline phases, with the contents of the latter ranging from a few wt.% to several hundredths or tenths of wt.%. The study of such materials using the method of X-ray phase analysis faces difficulties, when determining the phase structure. In this work, we will develop a method for the analysis of the diffraction patterns of such materials, when diffraction patterns include X-ray lines, whose intensities are at the noise level. The identification of lines is based on the search for correlations between the experimental and test lines and the verification of the coincidence making use of statistical methods (computer statistics). The method is tested on the specimens of a-quartz, which are often used as standard ones, and applied to analyze lava-like fuel-containing materials from the destroyed Chornobyl NPP Unit 4. It is shown that the developed technique allows X-ray lines to be identified, if the contents of separate phases is not less than 0.1 wt.%. The method also significantly enhances a capability to determine the phase contents quantitatively on the basis of lines with low intensities.

D2Dplot: 2D X-ray diffraction data processing and analysis for through-the-substrate microdiffraction

The d2Dplot computer program provides a set of tools for the visualization, processing and analysis of 2D X-ray diffraction (2DXRD) data. Among the operations available there are the sum/subtraction of 2DXRD images, conversion to 1D data (powder pattern), azimuthal plotting, calibration of instrumental parameters, background subtraction and a command-line mode to run operations inside data processing pipelines. The graphical user interface allows easy use of the program. It also includes two main features: (i) the possibility of creating a user compound database to help in the fast phase identification of similar samples, and (ii) a detailed peak analysis routine for the application of the through-the-substrate microdiffraction methodology.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity

Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg(82) and Thr(201), linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg(82)-Thr(201) hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.

Duplication of Genes in an ATP-binding Cassette Transport System Increases Dynamic Range While Maintaining Ligand Specificity

Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This, however, creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein in a mannose ATP-binding cassette transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrates that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest that this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The x-ray crystal structures of the β-mannose-bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two periplasmic-binding proteins in ATP-binding cassette transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates.

Development of Hydrocracking Catalyst Support from Kaolin of Indonesian Origin

The global shift of petroleum refinery towards heavier crude oils means an increasing demand of hydrocracking catalysts. This work studies the conversion of a kaolin originating from the Bangka island in Indonesia into a hydrocracking catalyst support consisting of zeolite-Y and amorphous alumina-silica phases. After a beneficiation process combining controlled settling and adsorption of the kaolin suspension in water with the addition of 125 ppm of 0.01% polyacrylamide solution as flocculant and 156 ppm of 0.1% calcium chloride solution as the adsorbent, the kaolinite phase content is increased from 63.6 to 74.3 %-mass. After spray drying, the kaolin is calcined at three temperatures for 2 hours each, producing calcined kaolin phases K700C at 700 °C, K1013 at 1013 °C, and K1050C at 1050 °C. Temperatures of calcination for obtaining calcined kaolin phases is determined based on result of DSC/TGA. Synthesis of zeolite-Y is done by mixing varying proportions of these three calcined kaolin products, and zeolite-Y crystal seeds. These mixtures are aged at room temperature for 11 hours prior to reaction in a hydrothermal condition at 93 °C for 15-21 hours. The best calcined kaolin composition is found to be K700C : K1013C : K1050C = 10:85:5 (%-mass), resulting in a zeolite NaY purity of 86-88 % as characterized by X-ray diffraction (XRD), average ratio of SiO2/ Al2O3 of 5.35, mean pore diameter of 23.1 Å, specific surface area of 186 m2/g, and a total pore volume of 0.107 mL/g as measured by N2 adsorption. In a parallel manner, a series of amorphous silica-alumina (ASA) synthesis experiments is done to identify the best metakaolin calcination temperature and metakaoline activation pH. These are found to be 527 °C and 8.0, respectively, producing an ASA product with a 65 %-mass amorphous phase content as estimated by XRD data processing. To prepare the hydrocracking catalyst support, the zeolite NaY is added into the ASA-forming metakaolin sol at a pH of 8.0, aged for 8 hours at 50 °C. An initial morphological characterization of the obtained zeolite-ASA composite catalyst support suggests a good dispersion of the smaller zeolite NaY particles in the ASA microspheres.

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.

Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

Experience with exchange and archiving of raw data: comparison of data from two diffractometers and four software packages on a series of lysozyme crystals.

The International Union of Crystallography has for many years been advocating archiving of raw data to accompany structural papers. Recently, it initiated the formation of the Diffraction Data Deposition Working Group with the aim of developing standards for the representation of these data. A means of studying this issue is to submit exemplar publications with associated raw data and metadata. A recent study on the effects of dimethyl sulfoxide on the binding of cisplatin and carboplatin to histidine in 11 different lysozyme crystals from two diffractometers led to an investigation of the possible effects of the equipment and X-ray diffraction data processing software on the calculated occupancies and B factors of the bound Pt compounds. 35.3 Gb of data were transferred from Manchester to Utrecht to be processed with EVAL. A systematic comparison shows that the largest differences in the occupancies and B factors of the bound Pt compounds are due to the software, but the equipment also has a noticeable effect. A detailed description of and discussion on the availability of metadata is given. By making these raw diffraction data sets available via a local depository, it is possible for the diffraction community to make their own evaluation as they may wish.

Crystal Structure of a Voltage-gated K+ Channel Pore Module in a Closed State in Lipid Membranes

Voltage-gated K(+) channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K(+) channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 Å resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.