bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal Carbides & hydrides : Structure and Superconductivity under high pressure
Authors: Ahuja Rajeev
Published: November 2015

The long-standing prediction that hydrogen can assume a metallic state under high pressure, combined with arguments put forward more recently that this state might even be superconducting up to high temperatures, continues to spur tremendous research activities toward the experimental realization of metallic hydrogen. These efforts have however so far been impeded by the enormous challenges associated with the exceedingly large required pressure. Hydrogen-dense materials allow for the rather exciting opportunity to carry out a proxy study of metallic hydrogen and associated high-temperature superconductivity at pressures within the reach of current techniques. In the present work, we have used first-principles methods in an attempt to predict the superconducting critical temperature (Tc ) as a function of pressure (P) for metal-hydride systems. By comparing the obtained results, we are able to point out a general trend in the Tc-dependence on P. These gained insights presented here are likely to stimulate further theoretical & experimental studies of metallic phases of hydrogen-dense materials. CaC6 is superconducting with a critical temperature Tc of 11.5 K. Under pressure first Tc increases and then suppresses and the superconductivity of this compound is eventually destroyed at about 18 GPa. Here, we report a theoretical finding of the re-emergence of superconductivity in heavily compressed CaC6. The predicted phase III with carbon nanofoam is found to be stable at wide pressure range with a Tc up to 14.7 K at 78 GPa. For CaC2, we found that under compression, dumbbell carbon in CaC2 can be polymerized firstly into one-dimensional chain and then into ribbon and further into two-dimensional graphite sheet at higher pressure. Our results show that calcium can not only stabilize carbon sp2 hybridization at larger range of pressure but also contribute in superconducting behavior, which would further ignite experimental and theoretical interest.
Local structures of Zr in impact-related natural glass probed by XAFS
Authors: Tobase Tsubasa , Yoshiasa Akira , Wang Ling , Hongu Hidetomo , Hiratoko Tatsuya , Okube Maki
Published: November 2015

The local structures of tektites and natural glasses were studied by Zr K-edge XANES and EXAFS in order to provide quantitative data on bonding distances and coordination numbers. The XAFS measurements were performed at the beam line BL-NW10A of the PF-AR in National Laboratory for High Energy Physics (KEK), Tsukuba, Japan. Zr4+ ion in tektite has different kinds of coordination environment. Various natural glasses are formed under different physical conditions. Impact-related glass, fulgurite and volcanic glasses are typical natural glasses. Upon a devastating impact of a giant meteoroid on the Earth, particles of the Earth's surface were melted and catapulted into outer space, where they finally solidified and fell back to the Earth. Tektites should be formed by this series of processes [1]. Tektite has special local structure of Ca[2]. Glass structure is affected by the pressure and temperature conditions during the glass formation and quenching process. This study indicated that different formation process of natural glasses gives different local structure of zirconium ions. The Zr K-edge XANES spectra of tektite have the double post-edge peaks with different heights. The volcanic glasses and other impact-related glasses such as impactite possessed more simple XANES patterns. The average coordination number of Zr4+ in darwin glass, LDG, volcanic glass and tektite are between 6 and 7. The eight-coordinated Zr4+ shows different XAFS pattern in suevite and köfelsite. All tektites are classified in same type. According to EXAFS measurements, Zr-O distances in tektites are 2.198 – 2.215Å and XANES spectra of tektites have similar shape. It indicates that tektites have similar Zr local structure with 7-fold coordination Zr ions. Impact-related glasses are classified to different types. Volcanic glasses are classified to same types. Impact glasses are formed under different geological processes at impact event and are experienced different physical environments.
XAFS study on nanostructured (La;Sr)CoO and (La;Sr)(Co;Fe)O IT-SOFC cathodes
Authors: Mejía Gómez Augusto , Sacanell Joaquín , Soldati Analía , Fantini Marcia , Craievich Aldo , Figueroa Santiago , Lamas Diego
Published: November 2015

In the last years, extensive research has been devoted to develop novel materials and structures with high electrochemical performance for intermediate-temperatures solid-oxide fuel cells (IT-SOFCs) electrodes. In recent works, we have investigated the structural and electrochemical properties of nanostructured La0.6Sr0.4CoO3(LSCO) and La0.6Sr0.4(Co;Fe)O3(LSCFO) cathodes, finding that they exhibit excellent electrocatalytic properties for the oxygen reduction reaction [1,2]. These materials were prepared by a pore-wetting technique using polycarbonate porous membranes as templates. Two average pore sizes were used: 200 nm and 800 nm. Our scanning electronic microscopy (SEM) study showed that the lower pore size yielded nanorods, while nanotubes were obtained with the bigger pore size. All the samples were calcined at 10000C in order to produce materials with the desired perovskite-type crystal structure. In this work, we analyze the oxidation states of Co and Fe and the local atomic order of LSCO and LSCFO nanotubes and nanowires for various compositions by X-ray absorption spectroscopies. For this purpose we performed XANES and EXAFS studies on both Co and Fe K edges. These measurements were carried out at the D08B-XAFS2 beamline of the Brazilian Synchrotron Light Laboratory (LNLS). XANES spectroscopy showed that Co and Fe only change slightly their oxidation state upon Fe addition. Surprisingly, XANES results indicated that the content of oxygen vacancies is low, even though it is well-known that these materials are mixed ionic-electronic conductors. EXAFS results were consistent with those expected according to the rhombohedral crystal structure determined in previous X-ray powder diffraction investigations.
Studying the structural and electronic effects of substituted (Bi0.5Na0.5)TiO3
Authors: Blanchard Peter , Kennedy Brendan , Ling Chris
Published: November 2015

Significant efforts have been made in the development of (Bi0.5Na0.5)TiO3 ferroelectrics as an alternative to the lead-based industry standard PbTi1-xZrxO3.[1] It has also been shown that doping the A- and B-site of (Bi0.5Na0.5)TiO3 can greatly improve the ferroelectric behavior of these materials,[2] possibly due to the formation of two or more ferroelectric phases at a morphotropic phase boundary (MPB). As such, there is a significant interest in understanding the structural changes in (Bi0.5Na0.5)TiO3-based solid solutions. (Bi0.5Na0.5)TiO3 was originally described as adopting a rhombohedral structure in space group R3c, However, the accuracy of this description has been greatly debated. It was recently suggested that (Bi0.5Na0.5)TiO3 actually adopts a monoclinic structure in space group Cc.[3] Given this recent controversy, we investigated the structural evolution of (Bi0.5Na0.5)TiO3-based solid solutions, particularly the (Bi0.5Na0.5)Ti1-xZrxO3 and (1-x)(Bi0.5Na0.5)TiO3–xBiFeO3 solid solutions., using both diffraction and spectroscopy techniques. Diffraction measurements on (Bi0.5Na0.5)TiO3 confirm that both monoclinic Cc and rhombohedral R3c phases are present at room temperature. Diffraction analysis showed that doping (Bi0.5Na0.5)TiO3 with a small amount of (Bi0.5Na0.5)ZrO3 and BiFeO3 can stabilizes the rhombohedral phase. The Ti/Fe K-edge and Zr L3-edge XANES spectra analysis was performed to determine the effects doping has on the local displacement of the B-site cations.
Determination of the local structure in metal-complexes by combining XAS and XES
Authors: Soldatov Mikhail , Lomachenko Kirill , Smolentsev Nikolay , Soldatov Alexander
Published: November 2015

Nanoscale local atomic structure determines most of unique properties of novel materials without long range order. To study its fine details one has to use both computer nanodesign and advanced experimental methods for nanodiagnostics. The status of modern theoretical analysis of the experimental x-ray absorption spectra to extract structural parameters is presented. Novel in-situ technique for nanodiagnostics - extracting of 3D structure parameters on the basis of advanced quantitative analysis of X-ray absorption near edge structure (XANES) - has been developed. The possibility to extract information on bond angles and bond-lengths (with accuracy up to 0.002 nm) is demonstrated and it opens new perspectives of quantitative XANES analysis as a 3D local structure probe for any type of materials without long range order in atoms positions (all nanostructured materials and free clusters belong to this class of materials). Even more possibilities are opening by using simultaneously several experimental synchrotron based techniques: XANES and XES and/or RIXS. In the framework of these approaches, the results of recent studies of local atomic structure for several types of nanostructures including nanoclusters with different types of chemical bonding, core-shell nanoneedles and thin films of dilute magnetic semiconductors, 5d-transition metal-organic complexes, Cu1+ and Cu2+ binding sites in amyloid-β peptide, Co aqua complexes in aqueous solution, nanostructured materials for hydrogen storage and nanocatalysts based on zeolites and MOF are reported. Along with the calculations of conventional XANES and XES, we show a possibility to simulate core-to-core and valence-to-core RIXS as well. Molecular orbitals (or DOS) of metal complexes can be directly related to the peaks in XES spectra in RIXS maps. This information is essential for understanding of electronic structure of metal complexes and design of novel materials.
XAS, XES and DFT simulations to bridge local and macroscopic properties in GaAsN
Authors: Amidani Lucia , Ciatto Gianluca , Boscherini Federico , Filippone Francesco , Mattioli Giuseppe , Alippi Paola , Bondino Federica , Polimeni Antonio , Capizzi Mario , Amore Bonapasta Aldo
Published: November 2015

Although it is well known that dilute species often significantly modify the properties of solids in which they are hosted, a basic question remains open: how critical are their local properties in determining the macroscopic ones of the host material? Here, we address this issue by taking N in dilute III-V-N alloys as a paradigmatic case and propose an original approach based on synergic use of x-ray spectroscopies (XANES and valence – to – core XES at the N K-edge), density functional theory simulations and hydrogen exposure as a tool to modify N local bonding. Our results disclose the key role of the local N – Ga interactions in determining both N-related band states and the macroscopic effects of dilute N on the GaAs energy gap. The success of our approach indicates a new way to connect local and macroscopic properties.
Progress and challenges in the theory and interpretation of x-ray spectra
Authors: Rehr John , Kas Joshua
Published: November 2015

There has been dramatic progress in recent years both in calculations and the interpretation of various x-ray spectra, ranging from extended x-ray absorption fine structure (EXAFS) and diffraction-anomalous fine structure (DAFS) to near-edge structure (XANES) and inelastic x-ray scattering (IXS). Using synchrotron radiation x- ray sources, these spectroscopies have become powerful probes of complex materials ranging from catalysts and minerals to bio-structures and aqueous systems. Together with advances in analysis techniques, these methods permit an interpretation of spectra in terms of structural, electronic, magnetic and vibrational properties. We summarize these advances first with a heuristic description of the real-space approach used in the electronic structure and spectroscopy codes developed by our group [1]. Our approach is based on real-space multiple-scattering Green's function techniques, rather than wave-functions. This simplifies calculations of excited states and x-ray spectra, particularly the inclusion of key many-body effects and relativistic corrections. The approach is illustrated with applications to various x-ray spectra of complex materials. For example, DAFS takes advantage of the fine structure in the intensity of Bragg diffraction peaks near an absorption edge, and provides unique information that combines EXAFS and XRD experiment. We also discuss some recent theoretical developments leading to a new generation of codes including FEFF9 [2] and extensions for treating strongly correlated systems.
A comparative analysis on x-ray structure of cobalamin binding proteins
Authors: Sukumar Narayanasami
Published: November 2015

Vitamin B12 (Cobalamin;Cbl;B12; Figure) is a water soluble vitamin and is an essential component for the growth and development of many eukaryotes and prokaryotes organisms. B12 coordinates a cobalt ion in the center of the ring through the four pyrrole nitrogen atoms, the fifth ligand is dimethylbenzimidazole (DMB) moiety and the sixth ligand can be methyl group, deoxyadenosyl, cyanide or OH-. The structurally diverse groups of B12 binding proteins involved in various important biological functions. B12 adopts either base-on or base-off conformation in B12 binding proteins (Figure). An in-depth analysis on these structures was carried out using PDB coordinates (www.pdb.org) of a carefully chosen database of B12 binding proteins to correlate the overall folding of the molecule with phylogeny, the B12 interactions, and with biological function. The chosen database can be divided into three distinct groups. The first group is B12 transport proteins in mammals and the second is B12 transport proteins in E.Coli. The third group can be broadly clarified as B12-dependent enzymes. Results: The molecular architecture of the B12 binding proteins is diverse. It varies from a two-domain to multi-domain proteins. An analysis on the environment around B12 molecule shows that hydrogen bonds or vander waals interactions are dominant interactions between B12 and protein. Both conventional hydrogen bonds (N-H....X and O-H....X) and weak C-H....X hydrogen bonds play important role in these interactions. The number of protein residues interacting with B12 varies widely from 2 to 18 residues, depending on the nature of biological function. The analysis clearly establishes that B12 is amazingly adoptive to wide range of environments, namely polar, non-polar or charged. The B12 uses its functional groups both at head, corrin ring and in tail region (phosphate or DMB groups) at optimum level to form stable complex with partner proteins. Details will be presented.
Local dynamics of proteins and DNA evaluated from crystallographic B-factors
Authors: Schneider Bohdan , Gelly Jean-Christophe , de Brevern Alexandre , Černý Jiří
Published: November 2015

Temperature displacement factors also known as B-factors provide information about local dynamics of atoms. Because dynamic behavior of biomolecules is as important as their static molecular structures we decided to analyze B-factors in almost a thousand non-redundant crystal structures of protein-DNA complexes from a well-curated dataset described in Schneider et al. Nucleic Acids Research, 42 (2014). Biopolymer residues, amino acids and nucleotides, were classified according to their molecular neighborhood as solvent-accessible, solvent-inaccessible (buried, i. e. forming the protein core), or lying at protein-protein or protein-DNA interfaces. In addition, water molecules were labeled as solely bound to the biopolymer surface as the first hydration shell or as water bridges binding protein and DNA molecules. Distributions of scaled B-factors for these types of residues confirmed several expected features of protein and DNA dynamics but they also revealed some surprising facts. Solvent-accessible amino acids have B-factors larger than residues at both biomolecular interfaces and amino acids forming protein core are restricted in their movement the most. A really unique feature of the buried amino acids is that their side chains are restricted in their movements more than the main chains. Protein interior is therefore packed significantly better than protein-protein or protein-DNA interfaces. Low values of B-factors of the waters bridging protein and DNA molecules contrast with extremely high values of DNA phosphates. Characteristic features distinguishing different types of residues quickly vanish in structures with lower resolution and some of the observed trends are a likely consequence of improper refinement protocols that may need rectifying. Acknowledgments. This study was supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF and by grant P305/12/1801 from the Czech Science Foundation.
Brazzein and structurally similar proteins: structural/functional comparisons
Authors: Nagata Koji , Hongo Nobuko , Kameda Yasuhiro , Yamamura Akihiro , Sasaki Hiroshi , Lee Woo Cheol , Ishikawa Kohki , Suzuki Ei-ichiro , Tanokura Masaru
Published: November 2015

Brazzein, a 6.5-kDa protein consisting of 54 amino acids and four disulfide bonds, is the smallest sweet-tasting protein yet isolated from the wild African plant Pentadiplandra brazzeana. Brazzein has various desirable properties for use as a low-calorie sweetener in the diets of individuals suffering from diabetes, obesity, and metabolic syndrome. For example, brazzein has a high water solubility and a high thermostability. In addition, brazzein is 2000-times sweeter than sucrose on a weight basis. Both the solution and crystal structures of brazzein have been reported. In the crystal structure [1], brazzein has a defensin-like fold containing two α-helices and a three-stranded antiparallel β-sheet. Defensins are small cysteine-rich cationic proteins found in both animals and plants, which function by binding to the microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients. In fact, Yount and Yeaman reported that brazzein has antimicrobial activity against Gram positive (Bacillus subtilis and Staphylococcus aureus) and negative (Escherichia coli) bacteria and a fungus (Candida albicans) at pH 7.5 rather than pH 5.5 [2]. A search for proteins with a similar backbone fold to brazzein using the DALI server shows that structurally similar proteins to brazzein include plant defensins, scorpion neurotoxins (K+ channel blockers), arthropod defensins, mollusc defensins, mold defensins, and a plant trypsin inhibitor. These proteins commonly have a γ-core sequence. Here we compare their sequences, structures and functions, which has led to a conclusion that the C-terminal half of brazzein is important for its antimicrobial activity, brazzein will not have a neurotoxin activity, and it will not act as a trypsin inhibitor.
Structural Characterization of Transcription Factor C/EBPbeta
Authors: Miller Maria
Published: November 2015

The basic region:leucine zipper (bZIP) DNA-binding protein, C/EBPbeta, plays a central role in many vital cellular processes, but is also implicated in tumorigenesis, tumor progression, as well as viral replication within cells. C/EBPbeta binds to specific DNA sites as homo- or hetero-dimers and interacts with other transcription factors to control the transcription of a number of eukaryotic genes. C/EBPbeta is an intrinsically repressed protein that is activated in response to growth factors. This study employs a variety of techniques such as sequence analysis, molecular modeling, X-ray crystallography, and mutagenesis to provide structural insights into the mechanisms that modulate the biological activities of C/EBPbeta. Analysis of the primary structure indicates that C/EBPbeta is a largely disordered protein that consists of unstructured regions that have the potential to fold upon binding to molecular partners as well as regions that retain irregular conformations regardless of their environment. Here, a model of the auto-inhibited form of C/EBPbeta is presented as well as the structural basis of its specific dimerization, DNA-binding, and interactions with the p300 transcriptional co-activator.
Structure-function characterisation of Chlamydophila pneumoniae MOMP
Authors: Atanu Frank , Danson Amy , Watson Kimberly
Published: November 2015

The major outer membrane protein (MOMP) from Chlamydophila pneumoniae is a promising candidate antigen for chlamydophila vaccine development. MOMP is a 40kDa protein, encoded by the gene omp1, accountable for 60% of the total outer membrane mass of Chlamydophila pneumoniae. 2-3% of all Gram negative genomes encode for this particular protein class (porins), emphasising its importance and fueling intensive research into MOMPs structure and function. Our particular interest is in the established link between human infection, by micro-organisms such as Chlamydophila pneumoniae, and atherosclerosis – a multifactorial killer disease in developed nations. Evidence suggests a role for purified MOMP and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE–/– mice through dampening of MHC class II activity. We have used bioinformatics, SRCD and FTIR spectroscopies, and electrophysiology to reveal details of the structure, stability and function of MOMP. Our research to date demonstrates MOMP as a beta-barrel membrane protein containing putative 'hatch' and 'plug' domain helices, which may have implications in its function as a porin. Additionally, we show that MOMP exhibits significant increased thermal stability in the presence of fatty acids, highlighting a role for key 'NPA' and 'NPS' signature motifs present in MOMP in the transport of solutes. Our results proffer solutions to the long standing bottleneck in recombinant production of Chlamydophila MOMPs and their functional characterisation. This work has promising implications for structure-driven vaccine design against Chlamydial related diseases.
Structures of bovine cytochrome oxidase reveal proton active transports
Authors: Yano Naomine , Shinzawa-Itoh Kyoko , Shimada Atsuhiro , Takemutra Shuhei , Kawahara Takako , Tadehara Hitomi , Nagao Shuuhei , Baba Junpei , Yoshikawa Shinya , Tsukihara Tomitake , Muramoto Kazumasa , Yamashita Eiki
Published: November 2015

Bovine cytochrome c oxidase (CcO) pumps four protons in each catalytic cycle through H-pathway including a hydrogen-bond network and a water channel in tandem. Protons, transferred through the water-channel from the negative side of mitochondrial inner membrane into the hydrogen-bond network, are pumped to the positive side of the membrane electrostatically by net positive charges on a heme (heme a) iron created upon electron transfer to the O2 reduction site. For blockage of backward proton leak from the hydrogen-bond network, which determines the proton-pumping direction, the water channel is closed after O2 binding to initiate proton-pump. Thus, four protons must be collected in the hydrogen-bond network before O2 binding. The X-ray structural analyses of the oxidized/reduced CcO at 1.5/1.6 Å resolution reveal a large cluster composed of ~21 water molecules and a Mg2+ site including Glu198 (Subunit II) bridging CuA and Mg2+. The cluster of the oxidized state have 20 water sites with full occupancy and two sites with partial occupies of water, while that of the reduced state have 19 water sites with full occupancy and 3 sites with partial occupancies. The carboxyl group of Glu198 changes its coordination structure to Mg2+ upon the reduction of the active centers. The cluster is tightly sealed sterically against proton exchanges with the cluster outside except for a short hydrogen-bond network connecting the cluster with H-pathway. Five proton-acceptable groups hydrogen-bonded with the cluster suggest sufficient storage capacity for four proton equivalents. The redox-coupled structural changes in the electron transfer pathway from CuA, the initial electron acceptor from cytochrome c, to heme a suggest redox-driven effective proton donations from the cluster to H-pathway, facilitated by Glu198. These results indicate that the cluster is a crucial element of the proton-pumping system of bovine CcO.
The trans-membrane 17βHSD7: Kinetic study and preliminary crystallization data
Authors: Theriault Jean-Francois , Zhu Dao-Wei , Lin Sheng-Xiang
Published: November 2015

Human 17β-Hydroxysteroid dehydrogenase type 7 (h17HSD7) is a transmembrane enzyme belonging to the large, phylogenetically related protein family of short-chain dehydrogenases/reductases (SDR). In this study, h17HSD7 was expressed, purified, and the steady-state kinetics was studied. Here we report a purification method of the transmembrane enzyme using FPLC equipment with a combination of detergents to obtain homogeneous protein. We also report steady-state kinetics for h17HSD7 at 37 degrees Celsius and pH 7.5, using a homogeneous enzyme preparation with estrone (E1), or dihydrotestosterone (DHT) as substrate and NADPH as the cofactor. Kinetic studies made over a wide range of concentrations of both steroids (2microM to 60microM) revealed that E1 (Km= 12microM) has a higher apparent affinity for the h17BHSD7 compared to DHT (Km= 44microM). Crystallization of h17HSD7 in complex with high concentration of steroids to saturate the enzyme in the drop as low apparent affinity of both steroids was found in kinetic study. Some preliminary crystals were obtained by hanging-drop and diffracted at synchrotron facility (Maximum resolution: 2.95Å , Space group: H 3 2, Average unit cell: 120.74 120.74 201.94 90 90 90, Mosaicity : 0.51).
X-ray crystal structure of voltage-gated proton channel
Authors: Takeshita Kohei , Sakata Souhei , Yamashita Eiki , Fujiwara Yuichiro , Okamura Yasushi , Nakagawa Atsushi
Published: November 2015

The voltage-gated proton channel, Hv1 (VSOP) has a voltage-sensor domain (VSD) but lacks an authentic pore domain, and the VSD of Hv1 plays dual roles of voltage sensing and proton permeation. Hv1 is required for high-level superoxide production by phagocytes through its tight functional coupling with NADPH oxidase to eliminate pathogens. Hv1 is also expressed in human sperm and has been suggested to regulate motility through activating pH-sensitive calcium channels. The activities of Hv1 also have pathological implications, such as exacerbation of ischemic brain damage and progression of cancer. In this study, our crystal structure of mouse Hv1 (mHv1) showed a "closed umbrella" shape with a long helix consisting of the cytoplasmic coiled-coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. We also found a Zn2+ion at the extracellular region of mHv1 protomer. The binding of Zn2+strongly suggested that the crystal structure of mHv1 represents the resting state, since Zn2+specifically inhibits activities of voltage-gated proton channels. Actually, two out of three arginines as sensor residues (R204 and R207) were located lower than the conserved phenylalanine, F146, on the S2 in a charge transfer center. This makes contrast with previous structures of other VSDs in the activated state where many positive residues of S4 were located upper than the conserved phenylalanine. Additionally, the crystal structure of mHv1 highlighted two hydrophobic barriers. Aspartic acid (D108), which is critical for proton selective permeation, was located facing intracellular vestibule below the inner hydrophobic barrier, thereby being accessible to water from the cytoplasm. Another hydrophobic layer of extracellular side probably ensures interruption of the proton pathway of mHv1 in resting state. These findings provide a novel platform for understanding the general principles of voltage sensing and proton permeation.
Inward-facing conformation of the bacterial heme transporter
Authors: Sugimoto Hiroshi , Naoe Youichi , Nakamura Nozomi , Doi Akihiro , Shiro Yoshitsugu
Published: November 2015

Iron is an essential element for almost all organisms, since iron serves as a catalytic center for redox reactions in many enzymes. Bacterial pathogens need to acquire iron from tissues of host to survive. Heme transport by ATP-binding cassette (ABC) transporter plays a key role in pathological processes. In gram-negative bacteria, the heme or heme protein binds to specific outer membrane receptors on the bacterial surface. The heme is then transported into the cell via ABC transporters. Here, we present the crystal structure of the heme transporter complex BhuUV-T from Burkholderia cenocepacia at 3.5 Å resolution in nucleotide-free state. The permeation pathway created by transmembrane helices of two BhuU subunit exhibits an inward-facing conformation. Comparison with the outward-facing conformation previously reported for the heme transporter HmuUV from Yersinia pestis and homologous vitamin B12 transporter BtuCD-F from E. coli indicates the structural mechanism involving the translational shift of nucleotide binding subunit and repositioning of the helices of permease subunits for substrate translocation. Structure of interface between BhuUV and periplasmic heme-binding protein BhuT suggests that the acidic residues of BhuU at the periplasmic interface may have an important role in releasing the heme from BhuT. We also determined the BhuT in apo and two types of holo form, providing the structural basis for transient and ambiguous heme recognition.
Biophysical characterization and crystallization of the membrane protein Etk
Authors: Prince Chelsy , Jia Zongchao
Published: November 2015

The inner membrane protein E. coli tyrosine kinase (Etk) is part of a large protein complex that assembles and exports capsular polysaccharide (CPS) in Gram-negative bacteria. Etk interacts with an outer membrane protein channel, YccZ (1), and regulates CPS export through autophosphorylation of a tyrosine cluster in its C-terminal tail. Previous work resulted in the structure of the isolated Etk C-terminal kinase domain (2). In the present study, the full-length protein has been purified and characterized in vitro. When purified in n-dodecyl-β-D-maltoside (DDM), Etk full length retains autophosphorylation activity, but is not suitable for crystallization because it severely aggregates and degrades. Using the main degradation product, a truncation containing the N-terminal domain (interacts with YccZ) and both transmembrane helices was designed. Truncated Etk does not further degrade and exists as a mixture of monomers and dimers when solubilized by five detergents as determined by size-exclusion chromatography and analytical ultracentrifugation. Crystals have been successfully grown when the protein is solubilized in DDM or n-decyl-β-D-maltoside (DM). The most promising crystals (DDM, 0.1 M MES pH 6.0, 1-5% PEG 3000, 20-30% PEG 200) have been reproduced and optimized with the assistance of a colorimetric assay (3). This assay relies on a reaction between 2,6-dimethylphenol, sulfuric acid, and the sugar head group of certain detergents to accurately quantify detergent in crystallization samples with minimal sample loss. Additive screening also revealed that MgCl2 improves crystallization. Optimization of this crystallization condition has significantly improved reproducibility of these crystals, but x-ray diffraction is limited to 6.5 Å. Current work is focused on reproducing and optimizing a second crystallization lead (DM, 0.1 M KH2PO4 pH 7.5, 32% PEG 400, 0.1 M KCl).
Conformational change of periplasmic heme-binding protein in ABC transporter
Authors: Nakamura Nozomi , Naoe Yoichi , Doi Akihiro , Shiro Yoshitsugu , Sugimoto Hiroshi
Published: November 2015

Iron is one of the essential elements for all living organisms. Pathogenic bacteria acquire heme from the host proteins as an iron source. Gram-negative opportunistic pathogen, Burkholderia cenocepacia have ATP-binding cassette (ABC) transporter BhuUV-T complex to permeate heme through inner membrane. BhuT, periplasmic binding protein (PBP), bind and deliver heme(s) to inner membrane transporter BhuUV complex. BhuUV is 2:2 complex of the transmembrane permease subunit and cytoplasmic ATP-binding subunit which couple ATP hydrolysis to solute translocation. The molecular level mechanism of heme recognition and dissociation by PBP and heme transport by transporter complex are not fully understood. Here we describe the crystal structures of the heme-free and two types of heme-bound state of BhuT. These crystals were obtained in different crystallization conditions. Crystals diffracted to high resolution at SPring-8. BhuT is composed of two globular domains linked by a long a-helix. The transport ligand heme is bound between the two domains. A detailed structural comparison of the conformation of the domain and residues involved in the heme binding will be presented.
Elevator mechanism of aspartate (glutamate) transport across the membrane
Authors: Guskov Albert , Jensen Sonja , Rempel Stephan , Hänelt Inga , Slotboom Dirk
Published: November 2015

Archaeal homologues of human neuronal glutamate transporter catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions in the cytoplasm the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here we present a crystal structure of the substrate-free transporter GltTk from Thermococcus kodakarensis, resolved at 3 Å resolution [1]. Despite the global similarity to the previously resolved structures of aspartate transporter GltPh, there are tremendous rearrangements in the substrate-binding site. The key binding residue Arg401 moves in and partially occupies the substrate's position, while the rotation of another conserved residue Met314 completely destroys the geometry of the sodium-binding sites. This structure provides direct structural insight in the mechanism of the essential reorientation step in the translocation cycle for this type of transporters.
Structures of the human two-pore domain potassium channels TREK-1 and TREK-2
Authors: Pike Ashley , Dong Yin , Mackenzie Alexandra , McClenaghan Conor , Mukhopadhyay Shubhashish , Burgess-Brown Nicola , Tucker Stephen , Carpenter Elisabeth
Published: November 2015

TREK-1/2 are members of the mechano-gated subfamily of two-pore (K2P) domain potassium channels leaking K+ out of the cell and contributing to the resting membrane potential. In contrast to the classical tetrameric potassium channels, K2P channels are dimeric with an atypical architecture and the structural mechanisms underlying their channel gating are poorly understood. Here we present the crystal structures of human TREK-1 and TREK-2 at resolutions of 2.7 and 3.4Å which provide insights into the basis of intracellular and extracellular gating in this unique family of ion channels. We have solved the structure of TREK-2 in two distinct conformations differing in the orientation of the pore-lining transmembrane helices. The C-terminal M4 helix is hinged at a conserved glycine residue so that it adopts one of two distinct orientations. The M4 helix is either kinked towards the membrane, packing against the M2 inner helix of the adjacent subunit ("M4 up") or straightens and interacts with the M2/M3 helices from the same subunit ("M4 down"). In the M4 down state, a hydrophobic lateral opening runs perpendicular to the ion conductance pathway between M2 and M4 and links the inner vestibule to the membrane-exposed face of the channel. Transition between the "M4 down" and "M4 up" conformations may play a role in channel activation and gating. Cocrystallisation with a TREK-1/2 channel inhibitor promotes the "M4 down" state. The structure of TREK-1 exhibits an "M4-up" conformation but is unusual in that the selectivity filter is significantly distorted with only two correctly-formed potassium sites. The structure also reveals a divalent ion binding site between the extracellular cap and the pore domain loop. The TREK-1 structure illustrates how changes at an extracellular site can affect the pore structure. The structures will be described in detail along with their implications for channel gating in response to intracellular and extracellular stimuli.
Crystal structure of the plant nitrate transporter NRT1.1
Authors: Newstead Simon , Parker Joanne
Published: November 2015

Nitrogen uptake and assimilation is a key limiting factor for plant growth and crop productivity and also acts a major signaling molecule, controlling many aspects of plant development. Many plants obtain nitrogen through the uptake of nitrate from the soil via specific membrane transporters. Two families of nitrate transporter have been identified that act within the root cell, termed NRT1 and NRT2. NRT1 members are predominantly low affinity transporters, with KM values in the millimolar range, whereas NRT2 members are high affinity transporters, with KM values in the low micromolar range. Dual affinity transporter systems are used in biology to allow the cell to respond to changes in an external nutrient supply. In the case of nitrate transport in plants, decreasing levels of external nitrate cause an increase in the expression of NRT2 family transporter genes, in particular NRT2.1, allowing the cell to take up more of the available nitrate. However, plants have evolved a faster way of responding to nitrate levels involving post-translational control of nitrate uptake. NRT1.1 also known as CHL1, is a dual affinity nitrate transporter. In response to decreasing levels of nitrate, NRT1.1 is capable of switching between low and high KM modes, a switch achieved through the post-translational phosphorylation of an intracellular threonine. Here I will present our recently determined crystal structures of NRT1.1 in both the apo and nitrate bound forms. Together with in vitro binding and transport data we identify key residues involved in nitrate recognition and provide the first biochemical explanation for the phosphorylation controlled `dual affinity' switch observed in NRT1.1. Finally we present our model for the molecular basis of nitrate uptake via this transporter.
Computational analysis of early stage crystallization based on UV fluorescence
Authors: Padayatti Pius , Petersen Max , Mullen Giles
Published: November 2015

Detection of initial crystal hits during de novo attempt of structure determination of a macromolecule is a critical step in the crystallization process. The most common strategy during the initial matrix screening is to identify a condition that can grow crystals readily. Such occurrences are rare events for difficult to crystallize samples, which led Rigaku Automation to focus on methods to identify microcrystalline precipitation and marginally interesting conditions for further expansion trials from a high throughput (HTP) environment. Usually, the initial crystals observed for tough to crystallize samples are microcrystalline and buried within precipitated samples. UV imaging is proven to be a powerful tool to analyze the microcrystalline nucleation in crystallization trials. To further analyze UV imaging results we developed FluorScore, a numerical method that reduces the UV image to a single numeric metric that can be used to categorize the drops in terms of their type and intensity of fluorescence signal. This study will analyze the usefulness of the algorithm for successful detection of promising conditions for tough to crystallize novel samples along with some standard proteins for comparison. An evaluation of the challenges of analyzing the results of crystallization trials by exhaustive manual inspection compared to FluorScore's algorithm results will be presented. In situations of HTP crystallization trials, the described algorithm is a very useful tool that can be used to identify conditions that harbor microcrystal nuclei and help Crystallographers make confident decisions on expansion trials.
How phase measurements can change X-ray crystallography
Authors: Morelhao Sergio , Amirkhanyan Zohrab , Remedios Claudio , Guzzo Cristiane
Published: November 2015

"From inorganic crystals to macromolecular crystals, structure determination with atomic resolution is based on diffraction techniques (electrons, X-rays and neutrons). However, since the coherent scattering cross-section for X-rays by atoms have intermediate values between those for electrons and neutrons, experimental measurements of the phases of structure factors are feasible only for X-rays. Unprecedented results [1-3] have show how phase measurements can reveal important information of the crystalline structure of doped crystals, which are inaccessible by other techniques: ""differently from any other method in X-ray crystallography based on structure refinement of intensity data, the presented method pinpoint a specific feature of the structure and directly prove its existence beyond of any reliability parameter or of goodness-of-fitting value."" Although there are instrumental and computational challenges, the method can be extended to protein crystallography for improving resolution of poorly solved features of the structures. As depicted in Fig. 1, the structure factor of a given reflection has contributions of distinct groups of atoms. Low resolution at one group can result in phase deviation of Fickle, without significantly affecting its amplitude. For instance, small differences in the symmetry of charge balance at group B can cause great difference in the phase angle of this group, which increases the phase angle of Fickle, Fig. 1 (b). On the other hand, reduction in the scattering amplitude of group E due to atomic disordering decreases the phase angle, Fig. 1 (c). In this presentation, we discuss the current understanding of this method, its perspectives, and importance of providing a tool for structural analysis of macromolecules that is able to go beyond the resolution achieved by the techniques actually in use."
Evidence vs Expectations: How to validate your ligand in a protein structure
Authors: Pozharski Edwin
Published: November 2015

Determination of a protein-ligand complex structure is essential in many areas of structural biology. Details of the interactions between protein and a small molecule ligand often represent major findings from a crystal structure. Thorough validation of interpretation of such structural data is particularly important given high expectation of confirming prior experimental findings regarding targeted protein-ligand interaction. Modern methods of ligand validation are discussed and illustrated.
Short to long range order:Phase transition of one liquid crystal molecule at HP
Authors: Lee Jey Jau , Kao Ching-Che
Published: November 2015

We present the high pressure phase transition behavior of a gold(I) –NHCs complex, in which the NHCs include one long N-alkyl substituent and one N-acetamido group, The amide group is an excellent hydrogen bonding motif to provide interaction force between molecule. The gold (I) – NHC series form metallogel in DMSO at room temperature. PXRD studies show that self-assembly of [Au(C16,amide-imy)2 ][NO3] forms a lamellar structure with tubular architecture around the metal ion head core. Through Coulombic, hydrogen bonding, and hydrophobic interactions between solvent and the amide group under ambient environment. In the high pressure PXRD experiment of [Au(C16,amide-imy)2][NO3] complex , the lamellar structure phase become less and less , but the long range order behavior start to reveal by bragg's Debye ring grown up in the 2-D diffraction pattern when the pressure increased, As pressure up to 2GPa, The indexing shown the long range order with 3-D symmetry by monoclinic Laue symmetry. This is the first example that phase transition from lyotropic liquid crystal gel phase to long range order solid phase. The inter-molecule interaction and structure will be presented in this report.
Unprecedented adsorption of molecular hydrogen in the porous hydride framework
Authors: Filinchuk Yaroslav , Tumanov Nikolay , Ban Voraksmy , Oh Hyunchul , Hirscher Michael , Richter Bo , Jensen Torben , Hudson Matthew , Brown Craig , Iles Gail , Jorgensen Scott
Published: November 2015

"Recently the first porous hydride, gamma-Mg(BH4)2, featuring so-called ""borohydride framework"" and capable to store reversibly guest species was discovered [1]. This example clearly shows that the covalently bound hydride anions, such as borohydride, can act as directional ligands, capable to form molecular and polynuclear complexes, as well as framework structures typically occurring in classical coordination chemistry. Various small molecules are reversibly absorbed in gamma-Mg(BH4)2. In this work we show that molecular hydrogen and nitrogen have different adsorption sites in gamma-Mg(BH4)2, leading to different capacities on saturation and to different H2 and N2 BET areas. Only up to 0.66 N2 molecules are adsorbed per Mg atom, but the saturation capacity is double for the smaller hydrogen molecule. Moreover, at higher pressures, the second hydride phase forms with unprecedented hydrogen content of ~22 weight % (!). The density of hydrogen adsorbed into the pores is much higher than in liquid hydrogen, having no analogues among other porous systems. On the technical side, we will illustrate how in-situ diffraction at neutron and synchrotron sources allows to follow adsorption isobars, aiming for extraction of isosteric heats of adsorption directly from diffraction data, as well as for clarifying the microscopic mechanisms in terms of guest-host and guest-guest interactions."
Chemical Selectivity in Diffraction by Statistical Analysis of in situ XRPD Data
Authors: Milanesio Marco , Palin Luca , Viterbo Davide , Caliandro Rocco , Urakawa Atsushi , van Beek Wouter , Chernyshov Dmitry
Published: November 2015

X-ray diffraction methods in general allow only a limited chemical selectivity. Structural information on a subset of atoms can be obtained by a modulation enhanced diffraction (MED) experiment, using a periodic stimulus supplied in situ on a crystal, while diffraction data are collected several times within a stimulus period. The data are then treated by statistical methods such as phase sensitive detection (PSD) and Principal component analysis (PCA) techniques. The application of PSD to diffraction has been proposed as a tool to extract crystallographic information on a subset of atoms [1], thus allowing to introduce selectivity in diffraction. Simulated and experimental PSD-MED powder data were produced by using a TS-1 zeolite as spectator, in which Xe, acting as active species, is adsorbed and desorbed in a periodically modulated mode. By first demodulating these data, MED allowed to obtain the powder diffraction pattern of the active subset, i.e. to obtain selectively the crystallographic information on Xe, by solving the crystal structure of the active species out of the zeolite framework. The "real world" experiments indicated that the PSD-MED approach has some limitations related to its theoretical assumptions. PCA is widely used in spectroscopic analyses and was recently applied to XRPD data by some of us [2]. PCA was exploited to evaluate the in situ XRPD data quality, to speed up the data analysis and data pre-treatment required by PSD and improve the extraction of the substructure information from MED data. It resulted that the first two components obtained by PCA are related to the 1- and 2-omega patterns from PSD. The two approaches (PCA and PSD) are finally compared from the viewpoint of their capacity of gathering information on the Xe substructure inside the zeolite channels and used in a synergic way to obtain the optimal data analysis efficiency.