bluuebunny/crossref_metadata_2025 huggingface dataset:
Promiscuous substrate catalysis in tagatose-bisphosphate aldolase.
Authors: Low-Kam Clotilde , Sygusch Jurgen
Published: November 2015

Tagatose-1,6-biphosphate (TBP) from Streptococcus pyogenes offers a fascinating opportunity to probe promiscuous substrate turnover in an enzyme. TBP aldolase can cleave, apart from its own substrate TBP, three other bisphosphorylated D-hexoses including fructose-1,6-bisphosphate (FBP). These four sugars are diastereoisomers and differ in stereochemistry at carbon 3 and at carbon 4 with respect to the configuration of their hydroxyl groups. We have determined high resolution structures of the native enzyme in complex with natural substrates, FBP and TBP, and two competitive inhibitors. Since the TBP aldolase crystals are catalytically active, covalent reaction intermediates of TBP aldolase in complex with substrates were trapped under acidic conditions to minimize turnover. High resolution structural analysis revealed a snapshot of both substrates covalently trapped in the active site as Schiff bases undergoing C-C bond cleavage. The structural data pointed to Glu164, by virtue of hydrogen bonding to the substrate C4-OH, as the active site residue responsible for the proton abstraction at the C4-OH that initiates substrate cleavage. The isosteric mutant enzyme Glu164Gln, virtually devoid of activity, supported this interpretation. The structures of the Glu164Gln mutant in complex with TBP, FBP and two competitive inhibitors were solved at high resolution and corroborated the expected C4-OH hydrogen bonding of ligands with the carboxyaminde amide of Gln164, indicating retention of catalytically competent active site architecture upon mutation. The structural studies were underpinned with pH-profile studies that support Glu164 as the residue responsible for nonspecific substrate cleavage catalytic mechanism. Furthermore the pH-profile of the Glu164Gln was significantly different from that of the native enzyme corroborating Glu164 acting as the conjugate base for proton abstraction. The enzyme thus uses the same catalytic mechanism to cleave both diastereoisomers FBP and TBP, while promiscuous substrate recognition appears to be a function of subtle differences in the active site architecture when compared to the active site of the highly specific FBP aldolase from rabbit muscle.
Structural basis for the histamine synthesis by human histidine decarboxylase
Authors: Komori Hirofumi , Nitta Yoko , Ueno Hiroshi , Higuchi Yoshiki
Published: November 2015

Histamine is a bioactive amine responsible for a variety of physiological reactions, including allergy, gastric acid secretion, and neurotransmission. In mammals, histamine production from histidine is catalyzed by histidine decarboxylase (HDC). Mammalian HDC is a pyridoxal 5'-phosphate (PLP)-dependent decarboxylase and belongs to the same family as mammalian glutamate decarboxylase (GAD) and mammalian aromatic L-amino acid decarboxylase (AroDC). The decarboxylases of this family function as homodimers and catalyze the formation of physiologically important amines like GABA and dopamine via decarboxylation of glutamate and DOPA, respectively. Despite high sequence homology, both AroDC and HDC react with different substrates. For example, AroDC catalyzes the decarboxylation of several aromatic L-amino acids, but has little activity on histidine. Although such differences are known, the substrate specificity of HDC has not been extensively studied because of the low levels of HDC in the body and the instability of recombinant HDC, even in a well-purified form. However, knowledge about the substrate specificity and decarboxylation mechanism of HDC is valuable from the viewpoint of drug development, as it could help lead to designing of novel drugs to prevent histamine biosynthesis. We have determined the crystal structure of human HDC in complex with inhibitors, histidine methyl ester (HME) and alpha-fluoromethyl histidine (FMH). These structures showed the detailed features of the PLP-inhibitor adduct (external aldimine) in the active site of HDC. These data provided insight into the molecular basis for substrate recognition among the PLP-dependent L-amino acid decarboxylases.
Inner Workings of the UvrA·UvrB DNA Damage Sensor during Bacterial Nucleotide Excision Repair
Authors: Jeruzalmi David
Published: November 2015

Efficient elimination of DNA lesions by the nucleotide excision repair (NER) pathway is critical for all organisms. In bacteria, the NER pathway is implemented by the successive action of three proteins, UvrA, UvrB and UvrC via a series of large and dynamic multi-protein complexes. A large number of studies have defined three major stages associated with the early steps of the NER pathway. In stage 1, a large (300-400 kDa) complex of the UvrA and UvrB proteins (AB) scans the genome to identify lesion-containing DNA. This process requires rapid binding and release of DNA; moreover, damage must be specifically recognized, and distinguished from native DNA, despite the fact that the relevant lesions induce widely different DNA structures. Once lesion-containing DNA has been located, it is stably bound by a dimeric form of UvrA within the AB complex (Stage 2). A major reorganization then occurs in which UvrA is lost from the ensemble, and concomitantly, UvrB becomes localized at the site of damage (Stage 3). Following these early stages, additional events lead to excision of the damage on one strand, and repair of the resulting single-stranded gap. Over the past few years, we have determined three structures of UvrA and the UvrA·UvrB complex. Our first structure of isolated UvrA revealed its overall architecture, its DNA binding surface, and the arrangement of its four-nucleotide binding sites. In the structure of the complete UvrA·UvrB damage sensor, a central UvrA dimer is flanked by two UvrB molecules, all linearly arrayed along a DNA path predicted by biochemical studies. DNA is predicted to bind to UvrA in the complex within a narrow and deep groove that is compatible with native duplex DNA only. In contrast, the shape of the corresponding surface in our prior structure of UvrA is wide and shallow, and appears compatible with various types of lesion-deformed DNA. These differences point to conformation switching between the two forms as a component of the genome-scanning phase of damage sensing. We also show that the highly conserved signature domain II of UvrA, which is adjacent to the proximal nucleotide-binding site, mediates a critical nexus of contacts to UvrB and to DNA. Moreover, in the novel UvrA conformer, the disposition of this domain is altered such that association with either UvrB or DNA is precluded. Concomitantly, nucleotide is uniquely absent from the proximal binding site. Thus, the signature domain II is implicated in an ATP-hydrolysis-dependent conformational change that detaches UvrA from both UvrB and DNA after initial damage recognition. The disposition and number of UvrB molecules in the AB complex, both unanticipated, suggest that once UvrA departs, UvrB localizes to the site of damage by helicase-mediated tracking along the DNA. Together these results permit a high-resolution model for the dynamics of early stages in NER.
Mycobacterial Trehalose Synthase as a potential drug target for tuberculosis
Authors: Caner Sami , Nguyen Nham , Aguda Adeleke , Zhang Ran , Pan Yuan , Withers Stephen , Brayer Gary
Published: November 2015

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose to trehalose in mycobacteria as one of three biosynthetic pathways to this non-reducing disaccharide. Given the importance of trehalose to survival of mycobacteria there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site cleft within which the intramolecular rearrangement can occur with minimal hydrolysis. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.
Structural insights into archeal-type SAHase from Thermotoga maritima
Authors: Olszynska Patrycja , Imierska Monika , Czyrko Justyna , Brzezinski Krzysztof
Published: November 2015

S-adenosyl-L-homocysteine hydrolase (SAHase) catalyzes the reversible breakdown of S-adenosyl-L-homocysteine (SAH) to adenosine (Ado) and homocysteine (Hcy). SAH is formed in methylation reactions that utilize S-adenosyl-L-methionine (SAM) as a methyl donor. By removing the SAH byproduct, SAHase serves as a regulator of SAM-dependent biological methylation reactions.[1] Thermotoga maritima is a thermophilic bacterium, but its genome carries a number of archeal genes as a consequence of massive gene transfers related to adaptation to the high-temperature environment.[2] sahh is one of many genes of archeal origin found in T. maritima. Crystals of recombinant SAHase from T. maritima in complex with adenosine were obtained by the hanging drop vapor diffusion method. The crystals are monoclinic, space group C2, with a = 120.4, b =105.5, c = 85.5 Å, β=108.80and diffract X-rays to 1.80 Å. The crystal contains two protein molecules in the asymmetric unit. The enzyme is active as a homotetramer with a molecular weight of about 180 kDa. The crystal contains two protomers in the asymmetric unit, which exist in both, open and closed conformations. The complete tetrameric enzyme molecule is generated in the crystal lattice through the operation of the crystallographic twofold axis. In contrast to other SAHase structures, only two subunits contain a tightly bound NAD+ cofactor, however their closed conformations exclude the possibility of substrate binding. The other two subunits are in open conformation and bind adenosine molecule in the cofactor binding site. Herein, lack of the cofactor molecule excludes the possibility of an enzymatic reaction. In contrast to other SAHases, the C-terminal domain from adjacent protomer does not participate in the binding of the NAD+. Results presented here indicate a different structural organization of archeal type SAHases.
Structure based protein engineering to confer selectivity of guanine deaminase
Authors: Bitra Aruna , Anand Ruchi
Published: November 2015

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.
Bacterial Effector Kinases
Authors: Grishin Andrey , Cherney Maia , Condos Tara , Barber Kathryn , Anderson Deborah , Phanse Sadhna , Babu Mohan , Shaw Gary , Cygler Miroslaw
Published: November 2015

Protein phosphorylation is one of the main signaling mechanisms in eukaryotic cells. Not surprisingly, pathogenic adopted this mechanism to interfere with signaling processes in the host cell. To this end pathogens evolved kinases that, in addition to other bacterial effector proteins, are injected into the host cell via a syringe-like type 3 (T3SS) or type 4 (T4SS) secretion systems. Kinases NleH1 and NleH2 from pathogenic E. coli, OspG from Shigella, SteC and SboH from Salmonella, LegK1-4 from Legionella and YspK and YpkA from Yersinia represent currently known effector kinases. Some of these kinases were likely derived from eukaryotes via horizontal gene transfer (SteC, LegK1-4, YpkA). Other kinases (NleH, OspG, SboH and YspK) have been so far identified only in the pathogenic bacteria. The structures of NleH and OspG proved that these kinases, which are half the size of an average human kinase, contain only a core kinase fold. These kinases lack the main regulatory element – the activation loop. The structure of NleH suggests that it has no activation mechanism since the apo-kinase domain adopts an active conformation and no change is observed on nucleotide binding. The OspG kinase, which also contains only the core kinase fold, is stimulated by its binding partner, the ubiquitin-conjugating enzyme E2-ubiquitin complex. The structure of OspG:UbcH7-Ub complex shows that OspG binds the E2 and ubiquitin (Ub) at two distinct sites on its surface. In this complex the OspG active site is unobstructed and primed for catalysis. However the mechanism of OspG activation remains presently unknown. Both NleH and OspG were found to inhibit the NF-kB pathway, however the substrates forOspG and NleH kinase activities are not yet known.
Structural characterization of Get3/Get4/Get5 involved in TA-protein targeting
Authors: Chang Yi-Wei , Lin Tai-Wen , Li Yi-Chuan , Huang Yu-Shan , Sun Yuh-Ju , Hsiao Chwan-Deng
Published: November 2015

During the biosynthesis of tail-anchored (TA) membrane proteins, their single C-terminal trans-membrane segment is inserted into the ER membrane for orientating the functional domain(s) towards the cytosolic side of the cell. The machinery responsible for this post-translational process has only recently come to light. In yeast, the proteins participating in TA protein insertion include Get1/Get2, Get3, Get4 and Get5. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4/Get5 complex (Get4/5) from Saccharomyces cerevisiae. We show that Get3 interacts with Get4/5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle X-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4/5 complex to form an extended conformation in solution.
High-pressure neutron beamline at J-PARC and applications to earth sciences
Authors: Kagi Hiroyuki
Published: November 2015

Construction of the high-pressure dedicated beamline, PLANET, in Japan Proton Accelerator Research Complex (J-PARC) has been completed in 2012 and scientific programs for general users have just started in 2014. The PLANET beamline has a focusing mirror for incident neutron and two 900detector banks. Each bank has 160 pieces of Position Sensitive Detectors (PSDs) filled with 3He gas. Each bank has a detector coverage of 9001110against the incident beam in the horizontal direction and 001350in the vertical direction. Radial collimators are attached in front of the detector banks to reduce the background. The instrumental resolution is 0.6% in Δd/d. The accessible d-spacing is normally 0.2–4.2 Å and is doubled in a double-frame setup. The power of the proton beam is around 300 kW and will be increased to 600 kW in 2014. The most characteristic feature of the PLANET beamline is the multi-anvil apparatus with six independently acting 500-tonne rams (6-axis press called "ATSUHIME"). Using ATSUHIME, we successfully observed neutron diffraction patterns of hydrous minerals at high pressure and high temperature without any contamination from sample-surrounding materials such as pressure transmitting media, anvils, and so on. This clearly shows that the incident slit and radial collimator installed in the beamline are very effective to obtain the diffractions under high pressure. We focus on pressure-responses on the structure of materials with hydrogen- bonding networks through neutron diffraction measurements at high pressure. These results will contribute to fundamental understanding of hydrous materials in the deep earth and icy material in the planets. In the presentation, our recent achievements from PLANET beamline will be reported.
Incommensurate magnetic ordering and magnetoelectric coupling of BiFeO3
Authors: Park Je-Geun
Published: November 2015

BiFeO3 is arguably one of the most interesting multiferroic materials with both ferroelectric and magnetic transition above room temperature. Moreover, it has one of the highest polarization values, near 90 microC/cm2. The other interest lies in the cycloid magnetic structure with an unusually long-period of 620 Å. One of the key questions when it comes to the fundamental understanding of BiFeO3 is how the structure evolves as a function of temperature, especially across the cycloid transition, and whether there is any structural evidence of the most sought-after magnetoelectric coupling. The other principal question concerns with a full spin Hamiltonian necessary to describe the cycloid structure and the other fascinating physical properties. In order to answer these two questions, we have carried out high-resolution structural and dynamics studies using both neutron and synchrotron facilities on powder and single crystal samples. In this talk, we will highlight the structural evidence of the magnetoelectric coupling and high-field quenching of the cycloid above 20 Tesla from high-precision neutron scattering studies [1a,b,c]. We will also present inelastic neutron scattering data, from which we succeeded in establishing the full spin Hamiltonian [2-3].
Chiroptical study on benzil crystal using G-HAUP
Authors: Nakagawa Kenta , Isikawa Kazuhiko , Asahi Toru
Published: November 2015

There has been a great interest in understanding chiroptical properties such as optical rotatory power (ORP) and circular dichroism (CD) in anisotropic materials because these properties provide valuable information on molecular/crystal structure. In particular, generation of chiroptical properties in crystals composed of achiral compounds is considered as one of the most intriguing topics in the field of crystal optics. Benzil (C6H5COCOC6H5), which consists of two phenyl rings and two carbonyl groups, is achiral in solution, but exhibits chirality in the crystalline state. In the crystalline state, the molecules are arranged in a helical form and are put along three-fold left-handed screw axis or right-handed one. The benzil crystal belongs to trigonal crystal system with non-centrosymmetric space group P3121 or P3221. Hence, the crystal symmetry allows the benzil crystal to exhibit ORP and CD and indeed it is often used as a chiral reference crystal in visible/ultraviolet region (S. Chandrasekhar, 1954; N. K. Chaudhuri & M. A. El-Sayed, 1967) as well as α-quartz and nickel sulphate hexahydrate crystals. Meanwhile, ORP and CD reflect spatial dispersion of a second-rank permittivity tensor that depends on not only frequency but also wave vector of an electromagnetic wave. Furthermore, the magnitude of ORP and CD usually vary with the direction of an incident light. It is therefore much important in chiral science to discuss the relationship between ORP and CD along the principal axes and the molecular arrangements along them, respectively. In the case of benzil crystal, it is expected to show different ORP and CD along the a and c axes because of its trigonal crystal system, D3. In this study, we attempt to measure the ORP and CD of benzil crystal in the direction of the c and a axes with the Generalized-High Accuracy Universal Polarimeter (G-HAUP)(M. Tanaka et al., 2012).
Exploring Charge Transfer Mechanism in Organic NLO (Polymorphic) Materials
Authors: Munshi Parthapratim , Jha Kunal
Published: November 2015

The understanding of nonlinear polarization mechanism and their relation to the structural characteristics of non-linear optical (NLO) materials have been utmost importance considering a surge in the information technology and industrial applications. Some investigations in search of NLO materials have been carried out on organic molecule scaffolds such as stilbene and chalcone having π-e-donor acceptor substituent which facilitates intermolecular charge transfer; leading to the generation of good frequency conversion materials. Few of such materials are known to exhibit polymorphic characters and most of which crystallize in non-centrosymmetric space group – necessity for a NLO material. It has been realized that the chirality, polymorphic character and NLO activity of the chalcone derivatives are mainly governed by both the substitutions at the para position and the position of sulfur atom in the thiophene ring of thienylchalcone. This presentation will highlight the preliminary results towards the analyses of electron density distributions on NLO active stilbene and chalcone derivatives and their polymorphic forms. Therefore, explore the charge transfer mechanism responsible for the variation of NLO activity across these systems and on their non-centrosymmetric polymorphic forms. Charge densities modelled using both multipole formalism (Hansen & Coppens, 1978) and X-ray constraint wavefunction fittings (Jayatilaka & Grimwood, 2001) will be considered for these studies.
QM/electrostatics scheme for predicting optical properties of molecular crystals
Authors: Champagne Benoît , Seidler Tomasz , Stadnicka Katarzyna
Published: November 2015

In this contribution it is shown that modest calculations combining first principles evaluations of the molecular properties with electrostatic interaction schemes to account for crystal environment are reliable for predicting and interpreting the experimentally-measured electric linear and second-order nonlinear optical susceptibilities within the experimental error bars. This is illustrated by considering two molecular crystals, namely: 2-methyl-4-nitroaniline (MNA) and 4-(N,N-dimethylamino)-3-acatamidonitrobenzene (DAN) [1]. A good agreement between theory and experiment (see figure below for DAN) is achieved providing the electric field effects originating from the electric dipoles of the surrounding molecules are accounted for. The presentation will also i) highlight the key role of the geometry on the χ(1) and χ(2) responses, ii) demonstrate the impact of electron correlation on the molecular and crystal properties, iii) assess the performance of exchange-correlation functionals, and iv) address the amplitude of the zero-point vibrational energy contributions [2]. A second illustration will deal with the χ(1) and χ(2) responses of two anil crystals, [N-(4-hydroxy)-salicylidene-amino-4-(methylbenzoate) and N-(3,5-di-tert-butylsalicy-lidene)-4-aminopyridine, which can switch between a enol (E) and a keto (K) form [3].
Diffraction studies of Tavorite-based polyanionic materials for Li–ion batteries
Authors: Suard Emmanuelle , Bianchini Matteo , Ateba Mba Jean-Marcel , Masquelier Christian , Croguennec Laurence
Published: November 2015

Polyanionic materials attract great interest in the field of Li-ion batteries thanks to the wide range of possible available compositions, resulting in a great amount of different properties (1). For instance, the high working potential together with a capacity of 156 mAh/g (leading to a theoretical energy density of 655 Wh/g) made Tavorite LiVPO4F a widely studied material and a suitable candidate for commercial exploitation. Here we will focus our interest on the homeotype structure of LiVPO4O. This oxy-phosphate shows the ability to exploit two redox couples, V5+/V4+ at 3.95 V vs. Li+/Li and V4+/V3+ at an average potential of 2.3 V vs. Li+/Li upon Li+ extraction and insertion, respectively (2). The two domains show marked differences both in the electrochemical signature and in the phase diagram, which is extremely rich. In particular, while the high-voltage domain shows a relatively simple two-phase transformation between LiVPO4O and ε-VPO4O, the low-voltage domain is more complicated and it shows a series of three apparent biphasic reactions while Lithium is inserted in the Tavorite structural framework. To elucidate this reaction, we performed in-situ X-Ray diffraction (Kα1), i.e. we recorded the whole process in real time during battery discharge. The end member Li2VPO4O was also isolated ex-situ and its crystal structure determined for the first time thanks to neutron diffraction measurements (3). Both the phase diagram and the different crystal structures will be discussed.
In situ PDF and solid-state NMR studies of antimony anodes for Na-ion batteries
Authors: Allan Phoebe , Griffin John , Borkiewicz Olaf , Wiaderek Kamila , Darwiche Ali , Stratford Joshua , Chapman Karena , Chupas Peter , Monconduit Laure , Grey Clare
Published: November 2015

Sodium-ion batteries have attracted attention in recent years because of the natural abundance of sodium compared to lithium, making them particularly attractive in applications such as large-scale grid storage where low cost and sustainability, rather than light weight is the key issue [1]. Several materials have been suggested as cathodes but far fewer studies have been done on anode materials and, because of the reluctance of sodium to intercalate into graphite, the anode material of choice in commercial lithium-ion batteries, the anode represents a significant challenge to this technology. Materials which form alloys with sodium, particularly tin and antimony, have been suggested as anode materials; their ability to react with multiple sodium ions per metal-atom give potential for high gravimetric capacities[2]. However, relatively little is known about the reaction mechanism in the battery, primarily due to drastic reduction in crystallinity during (dis)charging conditions, but also because the structures formed on electrochemical cycling may not be alloys known to exist under ambient conditions. In this study, we present a study of antimony as an anode in sodium-ion batteries, using in situ pair distribution function (PDF) analysis combined with ex situ solid-state nuclear magnetic resonance studies. PDF experiments were performed at 11-ID-B, APS using the AMPIX electrochemical cell [3], cycling against sodium metal. Inclusion of diffuse scattering in analysis is able to circumvent some of the issues of crystallinity loss, and gain information about the local structure in all regions, independent of the presence of long-range order in the material. This approach has been used to probe local correlations in previously uncharacterised regions of the electrochemical profile and analyse phase progression over the full charge cycle. This analysis has been linked with ex situ 23Na solid-state NMR experiments to examine the local environment of the sodium; these show evidence of known NaxSb phases but indicate additional metastable phases may be present at partial discharge.
Time-resolved in situ neutron diffraction studies of Li-ion battery materials
Authors: Sharma Neeraj
Published: November 2015

Lithium-ion batteries are ubiquitous in society, used in everything from children's toys to mobile electronic devices, providing portable power solutions. There is a continuous drive for the improvement of these batteries to meet the demands of higher power devices and uses. A large proportion of the function of lithium-ion batteries arises from the electrodes, and these are in turn mediated by the atomic-scale perturbations or changes in the crystal structure during an electrochemical process (e.g. battery use). Therefore, a method to both understand battery function and propose ideas to improve their performance is to probe the electrode crystal structure evolution in situ while an electrochemical process is occurring inside a battery. Our work has utilized the benefits of in situ neutron diffraction (e.g. sensitivity towards lithium) to literally track the time-resolved evolution of lithium in electrode materials used in lithium-ion batteries (see Figure 1). With this knowledge we have been able to directly relate electrochemical properties such as capacity and differences in charge/discharge behaviour of a battery to the content and distribution of lithium in the electrode crystal structure. This talk will showcase some of our in situ investigations of materials in lithium-ion batteries, such as LiCoO2, LiFePO4, Li1+yMn2O4, LiNi0.5Mn1.5O4 and Li4Ti5O12/TiO2 electrodes. In addition, selected examples of our work using time-resolved in situ X-ray diffraction to probe other batteries types, such as primary lithium and secondary (rechargeable) sodium-ion batteries will be presented. Using time-resolved diffraction data, a comprehensive atomic-scale picture of battery functionality can be modelled and permutations can be made to the electrodes and electrochemical conditions to optimize battery performance. Therefore, crystallography and electrochemistry can mesh together to solve our energy needs.
Crystallographic studies on Mn atoms in Photosystem II using K-edge wavelength
Authors: Umena Yasufumi , Kawakami Keisuke , Shen Jian-Ren , Kamiya Nobuo
Published: November 2015

Molecular oxygen on Earth is generated from photosynthesis by cyanobacteria, algae and plants, where water molecules are split by Photosystem II (PSII). PSII catalyzes light-induced water oxidation leading to the production of protons, electrons and molecular oxygen. The catalytic center of oxygen evolving complex (OEC) in PSII is composed of four Mn atoms and one Ca atom organized in a Mn4CaO5-cluster, which cycles through several different redox states to accomplish the catalytic process. Cyanobacterial PSII is a multi-subunits membrane protein complex composed of 17 membrane-spanning subunits, 3 membrane-extrinsic subunits and about 80 co-factor molecules with a total molecular weight of 350 kDa as a monomer. We reported the PSII structure at 1.9 Å resolution prepared from Thermosynechococcus vulcanus (PDB code: 3ARC)[1]. We determined unambiguously the positions of the atoms in OEC using the electron density map corresponding to each of five metal atoms and five oxygen atoms, for the first time. However, the valences of each of the four Mn atoms and their participation in the redox reactions in OEC are not fully understood. In order to uncover the catalytic mechanism of light-induced water oxidation by OEC, it is important to determine the valence of each Mn atom as well as to solve the detailed structure. In this study, we analyze the electronic state of each Mn atom in OEC by X-ray crystallographic analysis using Mn K-absorption edge wavelength. The Mn K-absorption edge depends on the oxidation number, and the anomalous scattering factor changes greatly for the Mn atoms in different oxidation states. We collected the anomalous difference data from PSII crystals using the wavelength (~1.8921 Å) on the Mn K-absorption edge at beamline BL38B1 and BL41XU of SPring-8 in Japan. The calculated anomalous difference Fourier map indicated different intensities among the four Mn atoms in OEC. This may suggest the different electronic state among the four Mn atoms in OEC. However, there is a possibility that these Mn atoms are reduced by X-ray exposures to some extent, and so the valences of these Mn atoms were not determined completely. We will discuss the relationship between peak heights of the anomalous difference Fourier map and the valence among the four Mn atoms in OEC.
Rapid molecular replacement of coiled-coil and transmembrane proteins with AMPLE
Authors: Thomas Jens , Keegan Ronan , Bibby Jaclyn , Winn Martyn , Mayans Olga , Rigden Daniel
Published: November 2015

Molecular Replacement (MR) is an increasingly popular route to protein structure solution. AMPLE[1] is a software pipeline that uses either cheaply obtained ab inito protein models, or NMR structures to extend the scope of MR, allowing it to solve entirely novel protein structures in a completely automated pipeline on a standard desktop computer. AMPLE employs a cluster-and-truncate approach, combined with multiple modes of side chain treatment, to analyse the candidate models and extract the consensual features most likely to solve the structure. The search models generated in this way are screened by MrBump using Phaser and Molrep and correct solutions are detected using main chain tracing and phase modification with Shelxe. AMPLE proved capable of processing rapidly obtained ab initio structure predictions into successful search models and more recently proved effective in assembling NMR structures for MR[2]. Coiled-coil proteins are a distinct class of protein fold whose structure solution by MR is not typically straightforward. We show here that AMPLE can quickly and routinely solve most coiled-coil structures using ab initio predictions from Rosetta. The predictions are generally not globally accurate, but by encompassing different degrees of truncation of clustered models, AMPLE succeeds by sampling across a range of search models. These sometimes succeed through capturing locally well-modelled conformations, but often simply contain small helical units. Remarkably, the latter regularly succeed despite out-of-register placement and poor MR statistics. We demonstrate that single structures derived from successful ensembles perform less well, and comparable ideal helices solve few targets. Thus, both modelling of distortions from ideal helical geometry and the ensemble nature of the search models contribute to success. AMPLE is a framework applicable to any set of input structures in which variability is correlated with inaccuracy. We also present preliminary data demonstrating structure solution of transmembrane helical structures using Rosetta modelling. We finally consider future sources of starting models which offer the hope that MR with AMPLE, in the absence of close homology between a known structure and the target, may soon be possible with larger proteins.
Alternative phasing method in macromolecular crystallography
Authors: Panjikar Santosh , de Sanctis Daniele
Published: November 2015

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].
When protein crystal dehydration helps
Authors: Lobley Carina , Sanchez-Weatherby Juan , Sandy James , Mazzorana Marco , Krojer Tobias , Sorensen Thomas
Published: November 2015

A typical protein crystal contains 30-60% solvent. For a naked crystal, this solvent is distributed between solvent shells, where water and solvent molecules make specific interactions with the crystalline protein, and solvent channels filled with disordered solvent molecules. This internal solvent map of the crystal can be modified by placing the crystal in a dehydrating environment. This may in turn induce changes to the crystal lattice and affect mosaicity, resolution and quality of diffraction data. A dehydrating environment can be generated around a crystal in several ways with various degrees of precision and complexity. In this study we have used the HC1 device (Maatel) to mount crystals an air stream of known relative humidity – a precise yet hassle-free approach to altering crystal hydration. We set out to analyse a range of different crystals to establish usable protocols that will allow one to explore to crystal hydration space, either by preparing samples before synchrotron beamtime or by undertaking the experiments during beamtime. Our results, considered in the light of the literature surrounding crystal dehydration, provide guidance for when dehydration can help diffraction.
NE-CAT Crystallography Beamlines for Challenging Structural Biology Research
Authors: Kourinov Igor , Capel Malcolm , Banerjee Surajit , Murphy Frank , Neau David , Perry Kay , Rajashankar Kanagalaghatta , Schuermann Jonathan , Sukumar Narayanasami , Ealick Steven
Published: November 2015

The NorthEastern Collaborative Access Team (NE-CAT) focuses on the design and operation of synchrotron X-ray beamlines for the solution of technically challenging structural biology problems and provides an important resource for the national and international research community. Currently NE-CAT operates two undulator beamlines: 24ID-C - tunable in the energy range from 6 to 22keV and 24ID-E - not-tunable, but optimized for Se SAD experiments. Both beamlines are equipped with state-of-the-art instrumentation. MD2 microdiffractometers installed at both beamlines provide very clean beams down to 5 microns in diameter and are capable of visualizing micron-sized crystals. Large area detectors (ADSC Quantum 315 at 24ID-E beamline and Pilatus-6MF at 24ID-C beamline), not only provide the best diffraction data, but also make possible to resolve large unit cells. Both beamlines are equipped with ALS style automatic sample mounters. Locally developed software suite RAPD provides data collection strategies, quasi-real time data integration and scaling and simple automated MR/SAD pipeline through 384 core computing cluster. Users of the beamlines are supported by experienced resident crystallographers. To meet the needs of technically challenging crystallographic projects, cutting-edge hardware and software ideas are implemented. A summary of beamline capabilities, technology, scientific highlights and details of availability will be presented. Funding for NE-CAT is provided through P41 grant from the NIGMS and from the NE-CAT member institutions.
Backstage the RNA Polymerase I Structure: The Art of Crystallography
Authors: Moreno-Morcillo Maria , Taylor Nicholas , Gruene Tim , Legrand Pierre , Rashid Umar , Ruiz Federico , Steuerwald Ulrich , Mueller Christoph , Fernandez-Tornero Carlos
Published: November 2015

Life relies on the accurate cascade of protein production from the genomic code, the DNA, via RNA. RNA polymerase I (Pol I) is one of three types responsible for the transcription of DNA to RNA. It is responsible for the production of rRNA incorporated into the Ribosome. Without the extremely low error rate of this synthesis process life would not have evolved beyond the status of bacteria and amoebae. The X-ray structures of the 14-subunit yeast RNA polymerase I [1,2] provide insight into one of the cornerstones in this process of life. Altogether four different crystal structures at about 3 resolution are now available. The biological impact of the beautiful structure will be presented by C. F.-T. at this meeting. Yet, not only biologically, but also crystallographically this work presents a real masterpiece. Before the strucure could be published the limits of crystallographic expertise and technology had to be exploited. Interesting structures of large complexes seldomly diffract to a resolution where the convenience of automated methods can be used. Great care was required at every step. Some of the important ones will be explained in this presentation: In order to overcome the lack of non-crystallographic symmetry, multi-crystal averaging across several crystal forms was applied. A weak anomalous signal, not sufficient by itself to solve the structure, was crucial to climb out of a dead end sink directed by model bias when the structure was only 50 complete. The program mrtailor [3] was written to combine the information from multiple sequence alignment with structural data to minimise model bias from external restraints. Mrtailor is particularly well suited to work with multi-domain structures. The options available in refmac5 were tweaked in order to reveal the hidden feature in the lake of noise. This presentation gives insight into ``high-end'' crystallography beyond common textbook knowledge and how to deal with difficult cases.
High Brightness Beam for Microcrystallography at GM/CA@APS
Authors: Fischetti Robert , Xu Shenglan , Makarov Oleg , Stepanov Sergey , Ogata Craig , Sanisivili Ruslan , Venugopalan Nagarajan , Becker Michael , Smith Janet
Published: November 2015

GM/CA is a world leader in the development of microcrystallography capabilities for biological macromolecules. The combination of the GM/CA-developed quad-mini-beam collimator and advanced rastering and vector collect software tools have revolutionized microcrystallography. Recently, beamline 23ID-B was reconfigured by shifting the focusing optics 3.8 m downstream. This significantly increased the source demagnification and led to a 4-fold increase in the 5- and 10-micron beam intensity. Beamline 23ID-D is also being upgraded. A Pilatus3-6M with a 1.0 mm thick X-ray sensor was commissioned in January 2014 allowing shutterless data collection with high S/N. The detector specifications include 100 Hz frame rate, 10 MHz/pixel count rate, and high X-ray efficiency. The beamline optics and endstation are also being upgraded to provide a high intensity beam whose size can be variable rapidly in the range of 1 - 20 micron, a new air bearing goniometer with a sphere-of-confusion (SOC) of ~100 nm, a miniature sample XYZ stage that allows centering and scanning of a micron-sized crystal, and a new on-axis-visualization system that provides high resolution optical images of sample crystals. Plans are being developed to upgrade the Advance Photon Source storage ring with a Multi-Bend Achromat lattice. The source properties will be dramatically improved primarily by reducing the horizontal source size to be comparable to the vertical source size, resulting in a 2-3 orders of magnitude increase in source brightness. Both beamlines will be significantly improved by the source upgrade. Moreover the new microfocusing optics for 23ID-D will fully exploit the new source and could deliver a 500 nm (FWHM) beam with >2e13 photons/sec. This unprecedented flux density will provide new opportunities and challenges, and allow the study some of the most important problems in biology. Details of these developments will be presented.
ELECTRON DENSITY STUDIES ON THE REGIOSELECTIVITY OF DEPROTONATION REACTIONS
Authors: Van der Maelen Juan , Cabeza Javier
Published: November 2015

The C-alkyl groups of cationic triruthenium cluster complexes of the type [Ru3(µ-H)(µ-κ2N1,C2-EtnMemPyHk)(CO)10]+ (EtnMemPyHk represents a generic C-alkyl-N-methyl-pyrazium species) have been deprotonated to give kinetic products that contain unprecedented C-alkylidene derivatives and maintain the original edge-bridged decacarbonyl structure. When the starting complexes contain various C-alkyl groups, the selectivity of these deprotonation reactions is related to the atomic charges of the alkyl H atoms, as suggested by DFT/natural-bond orbital (NBO) calculations. Three additional electronic properties of the C-alkyl C-H bonds have also been found to correlate with the experimental regioselectivity since, in all cases, the deprotonated C-H bond has the smallest electron density at the bond critical point (bcp), the greatest Laplacian of the electron density at the bcp, and the greatest total energy density ratio at the bcp (computed by using the quantum theory of atoms in molecules, QTAIM). The kinetic decacarbonyl products evolve, under appropriate reaction conditions that depend upon the position of the C-alkylidene group in the heterocyclic ring, towards face-capped nonacarbonyl derivatives (thermodynamic products). Theoretical calculations support the proposal that the selectivity of these deprotonation reactions is primarily determined by the atomic charge of the alkyl H atoms: the higher the charge the easier the deprotonation when the starting complexes contain various C-alkyl groups. On the other hand, although QTAIM results have been obtained here only from theoretical electron densities for the above clusters, comparisons with local and integral topological parameters derived from both experimental and theoretical electron densities for the related triruthenium complex [Ru3(μ-H)2(μ3-MeImCH)(CO)9] (Me2Im = 1,3-dimethylimidazol-2-ylidene) may easily be made.