bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of L-ribose isomerase from Cellulomonas parahominis MB426
Authors: Terami Yuji , Yoshida Hiromi , Uechi Keiko , Takata Goro , Kamitori Shigehiro
Published: November 2015

Monosaccharides and their derivatives which hardly exist in nature are so-called "rare sugars". Rare sugars have significance not only in food industries but also pharmaceutical industries. We discovered a novel L-ribose isomerase from Cellulomonas parahominis (CpL-RbI, 249 amino acids), which catalyzes the reversible isomerization between L-ribose and L-ribulose, L-allose and L-psicose, and D-talose and D-tagatose. Since CpL-RbI has a broad substrate specificity, it is useful for the production of various rare sugars. To elucidate the molecular basis of unique enzymatic properties of CpL-RbI, we determined its crystal structure. The N-terminal His-tagged CpL-RbI overexpressed in Escherichia coli was purified using a nickel affinity column. Crystals of CpL-RbI were obtained from a reservoir solution of 0.1 M sodium acetate trihydrate (pH 4.6) with 3.9 M ammonium acetate, by a hanging-drop vapor-diffusion method at 293 K (Space group C2221, a = 76.8, b = 88.6, c = 152.3 Å). X-ray diffraction data were collected up to 2.10 Å resolution using a Rigaku R-AXIS VII on a RA-Micro7HF rotating anode generator (40 kV, 30 mA) at 100 K. The structure was solved by a molecular replacement method with a structure of Acinetobacter sp L-ribose isomerase (4NS7) as a search model, and refined to R-factor of 0.227. CpL-RbI had a cupin-type beta-barrel structure, and the catalytic site was found between two large beta-sheets with a bound metal ion (Fig. 1). There were two protein molecules in an asymmetric unit, forming a homo-dimer with a non-crystallographic 2-fold symmetry (Fig.1). Furthermore, the PISA server showed that two dimers in crystal were associated to form a stable tetramer. Complex structures with substrates, L-ribose, L-allose and L-psicose, were also successfully determined. We will discuss a broad substrate specificity and catalytic reaction mechanism of CpL-RbI based on its three-dimensional structure.
LipL41, a Hemin Binding Protein from Leptospira santarosai
Authors: Sun Yuh-Ju , Lin Ming-Hsing , Hsiao Chwan-Deng , Chang Yuan-Chih , Yang Chih-Wei
Published: November 2015

Leptospirosis, a widespread zoonotic disease, is caused by pathogen Leptospira. Outer membrane lipoprotein is the potential virulence factor of Leptospira. LipL41 is one of the major lipoprotein and highly conserved in Leptospira spp. Previous study suggests that LipL41 bears hemin binding ability and might have a possible role in iron regulation and storage. The hemin binding ability of LipL41 is determined with a Kd = 0.5910.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], are found in LipL41 as 140Cys-Ser and 220Cys-Pro. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might involve in the protein-protein interaction of the supramolecular assembly
Investigation of potential drug targets involved in antibiotic sensing and cell wall modification
Authors: Sobhanifar Solmaz , Worrall Liam , Gruninger Robert , Wasney Gregory , Strynadka Natalie
Published: November 2015

The emergence of an increasing number of broad-spectrum antibiotic resistant bacteria such as MRSA over the last decade has made the understanding of the mechanisms underlying resistance critical. Resistance very often involves enzymes associated with the cell wall, a common target of antibiotics. Here we investigate the structure and function of enzymes involved in antibiotic sensing and cell wall modification, while using a customized "cell-free" protein synthesis approach to obtain sufficient yields of promising but difficult-to-express antibacterial drug targets.
PROXIMA 2A: A New Micro-focus Beamline for Macromolecular Crystallography
Authors: Duran Denis , Le Couster Sebastien , Fox Gavin , Fourme Roger , Meijers Rob , Samama Jean-Pierre , Savko Martin , Shepard William
Published: November 2015

PROXIMA 2A is a new micro-focus and energy tunable beamline dedicated to biological macromolecular crystallography at Synchrotron SOLEIL. The beamline officially opened in March 2013, and its first year of user operation has yielded excellent results. The X-ray source is a powerful in-vacuum U24 undulator coupled to a cryo-cooled Si[111] channel-cut monochromator and a pair of focussing bimorph mirrors in Kirpatrick-Baez configuration. This combination delivers a photon flux of over 10**12 ph/s into a focal spot of 10 μm × 5 μm (H×V FWHM), which is tunable over 6 – 15 keV. The supports of the optical elements have been designed to minimise the effects of vibrations and thermal dilations on the X-ray beam position, which is stable to within 5 microns over a day. The experimental station consists of a high performance micro-diffractometer, a cryostream, an area detector (ADSC Q315r), and an X-ray fluorescence detector. The X-ray energies for MAD experiments are directly calibrated on the sample. A robot equipped with a large 9 uni-puck dewar (CATS Irelec) is available to users for the automated transfer and screening of cryo-cooled samples. The users launch their experiments via an MXCuBE interface [1], which permits the centering of the sample, collecting of diffraction images, recording of X-ray spectra and the transfer of samples. The X-ray diffraction data are of an excellent quality, and the users readily exploit the micro-focused X-rays to select the best zones of their crystals. The first year of results from users has yielded a variety of success stories including novel protein structures resolved from crystals as small as 5 microns, as well as those solved by SAD & MAD methods. The future perspectives include automated helical and grid scans, in situ plate screening and multi-crystal merging techniques.
Crystal structure of an alleged mannosylphosphate transferase Ktr6p at 3.06 Å
Authors: Rodionov Dmitry , Marks Daniella , Romero Pedro , Berghuis Albert
Published: November 2015

Ktr6p is an alleged mannosylphosphate transferase from yeast Golgi. It has been implicated in decorating both O-linked and N-lined glycans with mannosylphosphate in vivo. However, based on sequence similarity, Ktr6p belongs to GT15 family of α-1,2-mannosyltransferases. To address this disagreement, the soluble portion of Ktr6p was expressed in P. pastoris and purified by liquid chromatography. The purified protein, GDP-mannose and various acceptors were used in a number of direct and indirect activity assays, however, neither manosyltransferase nor mannosylphosphate transferase activity was detected. Ktr6p was crystallized in a number of PEG- containing conditions, but the crystals resisted all attempts at cryoprotection. Three crystals were used to collect a 3.06 Å resolution dataset on a home source at room temperature. The crystals belong to P 21 21 21 spacegroup with 2 molecules per asymmetric unit. The structure was solved by molecular replacement using a structure of Kre2p, a close homolog from GT15 family (40% sequence identity). The structure was refined to R/Rfree 16.1%/21.2% The overall structure of Ktr6p is very similar to the structure of Kre2p having less than 2 Å overall backbone RMSD. However even at 3 Å resolution the difference in the active site is striking. The guanine moiety binding pocked is occluded by a well-ordered loop making GDP-mannose binding impossible in this conformation. Several aminoacid substitutions in the Mn2+ coordinating environment suggest that Ktr6 does not depend on manganese for its postulated activity. These observations indicate that Ktr6p functions quite differently from Kre2p.
Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti
Authors: Mugo Andrew , Kobayashi Jun , Yamasaki Taiji , Mikami Bunzo , Yoshikane Yu , Yagi Toshiharu , Ohnishi Kouhei
Published: November 2015

Mesorhizobium loti MAFF303099, a nitrogen-fixing symbiotic bacterium, harbors degradation pathway I for pyridoxine (PN); a free form of vitamin B6. Pyridoxine 4-oxidase (PNOX), a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, is the first enzyme in the pathway. It catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal. PNOX with a C-terminal His6 tag was overexpressed in E.coli JM109 cells and purified with a Ni-NTA agarose column and a QA52 column. The tertiary structures of PNOX and a complex of PNOX with pyridoxamine (PM), which is a substrate analog, were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. The FAD interacts with the PNOX protein through a network of hydrogen bonds, which are mainly found in the ribose and pyrophosphate moieties of the FAD molecule. The surface structure of PNOX molecule showed that it had an opening socket for access of substrates. The opening was followed by a tunnel that was linked to the active site cavity. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of H460A and H462A mutant PNOXs were very low, and H460A/H462A double mutant PNOX exhibited no activity. His462 may act as a general base for abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase family shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases.
Scoliosis of backbone in Z-DNA nucleotides
Authors: Luo Zhipu , Dauter Miroslawa , Dauter Zbigniew
Published: November 2015

According to Wikipedia "scoliosis is a medical condition in which a person's spine is curved from side to side. Although it is a complex three-dimensional deformity, on an X-ray, viewed from the rear, the spine of an individual with scoliosis can resemble an `S' or a `?', rather than a straight line". This definition fits very well with the syndrome observed in many structures of Z-DNA nucleotides. In contrast to the A- and B-forms of DNA, where the sugar-phosphate backbone smoothly follows the right-handed helical line, in Z-DNA the backbone winds as a left-handed zig-zag pattern, repeating every two base pairs. On the basis of the first structures of Z-DNA it was observed that the phosphate groups can be rotated towards the outside of the helix in the ZI type or towards the inside in the ZII type of Z-DNA. A large number of crystal structures of Z-DNA oligomers are currently available in the Protein Data Bank, where both backbone types are observed, often co-existing as alternative, partially occupied conformations. At first it was postulated that the backbone conformation adopted by Z-DNA depends on the presence of metal cations and polyamines in the crystal structures, but later it was realized that the ZI and ZII conformations are not specific to the sequence or the interactions with metal ions [1]. A comparison of larger number of high-resolution Z-DNA structures reveals that the structures can adopt a range of backbone torsion angles between the two values representing the canonical ZI and ZII types [2].
Stacking Interactions mediate Recognition in Ribosome Inactivating Protein
Authors: Kaur Punit , Yamini Shavait , Kushwaha Gajraj , Sinha Mau , Sharma Sujata , Singh Tej
Published: November 2015

Ribosome Inactivating Proteins (RIPs) are capable of inhibiting protein synthesis by catalytically hydrolyzing at specific purine residues from the sarcin / ricin loop of large ribosomal RNA. There are two types of RIPs: type 1 - RIPs (RIP-1) are single polypeptide chain proteins while type 2 - RIPs contain two polypeptide chains which are covalently linked by a disulphide bond. Studies have indicated that stacking interactions play a dominant role in the ligand binding to RIPs. However, the structural basis of these interactions with RIPs as well as the nature of stack pairing and associated molecular mechanisms are not clearly understood. In order to examine the significance of these stacking interactions and the role of various aromatic residues involved, we have carried out the structural analysis of the complexes of RIP-1 from Momordica balsamina (MbRIP-1) and crystallized it with six different sugar molecules, ribose, fucose, glucose, fructose, maltose and lactose. The crystals belong to hexagonal space group H3 with approximate cell dimension of a = b = 130 Å, c = 40.5 Å. The r.m.s. deviation for the Calpha atoms in the complexed structures was found to be in the range of 0.5 Å to 0.9 Å. The aromatic rings of sugars are seen to be involved in stacking with aromatic rings of Tyr 70 and Tyr 111. The side chain torsion angles chi1 and chi2 of Tyr 70 in the complexes with ribose, fucose, glucose, fructose, maltose and lactose were found to be 64° / 161°, 166° / 86°, 6° / 172°, 70° / 167°, 64° / 161° and -75° / 178° respectively. The sugars are held firmly in the binding site of RIP-1 with the help of stacking interactions and hydrogen bonds. The results presented here have revealed the significance of aromatic stack pairing and delineated the stabilizing role of tyrosine residue in these interactions.
Structure of the homology domain of the DNA replication proteins Sld3/Treslin
Authors: Itou Hiroshi , Muramatsu Sachiko , Shirakihara Yasuo , Araki Hiroyuki
Published: November 2015

DNA replication in eukaryotic cells is tightly regulated so that each replication origin in chromosomal DNA fires just once in proper timing during a cell cycle. Activation of the replicative helicase at the origins is a key step of the regulation. This process is achieved by loading of Cdc45 and GINS onto the helicase core complex on the origins. Sld3 is a critical regulator for this process: Sld3 and Cdc45 form a complex, which associate with origins in a mutually dependent manner, and further phosphorylation of Sld3 by a cyclin-dependent kinase recruits GINS to the origins through the interaction with Dpb11. Sld3 is well conserved in fungi, and Treslin/Ticcr is identified as its functional counterpart in metazoans. Although Sld3 and Treslin do not show significant sequence similarity, recent bioinfomatic study showed that they share a homology region (Sld3/Treslin domain). Functional role of the domain has remained to be elusive, thus we set out for the structural study of the Sld3/Treslin domain of budding yeast Sld3. The crystal structure showed that the domain has rhombic shape, and there is a highly positively charged area on the molecular surface. Taken together with the preceding studies, the area is considered to be the interface to Cdc45. Shape and character of the interface were consistent with the structure model of Cdc45 proposed by SAXS and bioinfomatic studies. Our structural and functional analyses showed that the domain alone is enough to form stable complex with Cdc45, and the binding with Cdc45 is stabilized by structurally flexible arm. The arm was conserved, thus our binding model of Sld3 and Cdc45 was suggested to be common in Sld3 and Treslin.
Crystal structure of Rv2258c, a putative SAM-dependent methyltransferase
Authors: Im Ha Na , Suh Se Won
Published: November 2015

The Mycobacterium tuberculosis protein Rv2258c belongs to a large family of putative S-adenosylmethionine (SAM)-dependent methyltransferases in mycobacteria. As part of a laboratory-scale structural genomics project on conserved hypothetical proteins in pathogenic bacteria, we have determined the crystal structure of Rv2258c from M. tuberculosis H37Rv at 1.8 Å resolution. The crystals of apo Rv2258c belong to space group C2, with unit cell parameters a = 109.2 Å, b = 140.6 Å, c = 97.2 Å, and β = 98.50. Assuming that three monomers are present in the asymmetric unit, the Matthews parameter and the solvent fraction are 3.32 Å3 Da-1 and 63.0%, respectively. The crystal structure of Rv2258c has been solved by a combination of the molecular replacement and single wavelength anomalous diffraction technique, using platinum atoms as anomalous scattering centers and the coordinates of the RebM from Lechevalieria aerocolonigenes (PDB entry 3BUS) as the search model. In addition, two binary complex structures, Rv2258c-SAM and Rv2258c-S-adenosylhomocysteine (SAH), have been determined by molecular replacement using the model of apo Rv2258c.
Structural studies of NenD1, an argininosuccinate lyase involved in the biosynthesis of nenemycin
Authors: Hamel Jérémie , Murphy-Després Xavier , Chen Ruidong , Picard Marie-Ève , Duvignaud Jean-Baptiste , Zhang Changsheng , Shi Rong
Published: November 2015

Nenemycin is a newly discovered 31-membered cyclodepsipeptide antibiotic isolated from an aquatic Streptomyces. This cyclic molecule contains ester and peptide bonds and is biosynthesized by the combined action of multiple enzymes. Among those is NenD1, an argininosuccinate lyase involved in the synthesis of 2S,3R--2,3-diaminobutanoic acid (DABA), a modified amino acid later added to the cycle of the nenemycin biosynthesis. The structure of NenD1 has been determined at  2.7 Å, showing the homotetrameric arrangement of the enzyme. A new crystal form was obtained after reductive methylation of lysine residues and the resulted 2.9 Å structure reveals features not present in the original. The active site is located at the interface of three subunits and its architecture was compared with other similar enzymes. Our study provides further insights into the synthesis mechanism of the unusual amino acid.
Structural and biophysical characterization of human RNase 6
Authors: Gagné Donald , Couture Jean-François , Simonović Miljan , Doucet Nicolas
Published: November 2015

Human members of the RNase A superfamily include eight rapidly evolving homologous enzymes with varying ribonucleolytic activity. All eight canonical members are characterized by the presence of two histidines and a lysine forming the catalytic triad, in addition to strictly conserved cysteines involved in the formation of 3 or 4 disulfide bridges essential for structural integrity. Despite these architectural and catalytic similarities, human RNases are functionally diverse and their biological activities remain elusive. Apart from degrading RNA with varying degrees of efficiency, these structural homologues have acquired a variety of distinct biological functions, including anti-bactericidal, cytotoxic, angiogenic, immunosuppressive, anti-tumoral and/or anti-viral activities. Among human members of this vertebrate-specific family, RNase 6 and RNase 8 have never been structurally resolved. To better understand its biological function and to characterize its molecular interactions with RNA and/or potential unknown ligands, we present the three-dimensional structure of human RNase 6 in its apo form. While the enzyme shares similar structural features with other members of the RNase A superfamily, we emphasize interesting differences unique to RNase 6 that may be pertaining to its unique biological function. Additional NMR, CD and ITC biophysical characterization in presence of RNA ligands will also be presented.
Ubiquitin recognition by UBZ and UMI domains for DNA damage response
Authors: Toma Aya , Takahashi Tomio , Sato Yusuke , Goto-Ito Sakurako , Yamagata Atsushi , Fukai Shuya
Published: November 2015

Double-strand break (DSB) and interstrand crosslink (ICL) are serious damages in DNA. Responses to these DNA damages include ubiquitination of damaged chromatin and other substrates, which recruit protein complexes required for DNA repair. Therefore, many proteins involved in DNA damage response contain ubiquitin-binding modules. For instance, a ubiquitin ligase RNF168, which catalyzes K63-linked polyubiquitination of histone H2A, contains two types of ubiquitin binding motifs, MIU (motif interacting with ubiquitin) and UIM (UIM and MIU-related Ub-binding domain). FAAP20, which recruits Fanconi anemia proteins (crosslink-repair factors), contains a UBZ (ubiquitin-binding zinc finger) domain. To date, mechanisms for ubiquitin recognition by UMI and UBZ domains have remained unclear. In this study, we determined crystal structures of RNF168 UMI and FAAP20 UBZ in complex with ubiquitin at 1.9 Å resolutions, respectively. SPR analyses using UMI and UBZ mutants, which were designed to disrupt Ub binding, confirmed that the observed interactions between Ub and UMI or UBZ are critical for binding. Our structure and the accompanying in-vitro structure-based mutagenesis experiments reveal the structural basis of these important recognition events.
SusD-like protein from the human gut bacterium Bacteroides thetaiotaomicron
Authors: Chaudet Marcia , Rose David
Published: November 2015

Nutritional research is continually demonstrating the strong interaction between the human colon microbiota and a healthy digestive system 1. It has been shown that this intestinal microbial population can influence our state of health including our metabolism, nutrient production and absorption, and the development of our immune system2. These symbiotic organisms have an important role in the metabolism of human dietary carbohydrates and exist by utilizing these sugars, which are not readily digested by upstream human enzymatic mechanisms. The ability of these microbes to utilize these sugars can also impact digestive disease states that include obesity, irritable bowel disorder, colonic cancer and Type 2 diabetes3. A dominant member of this environment is the bacterium Bacteroides thetaiotaomicron and has been characterized at efficiently utilizing carbohydrates in the colon. This symbiont has been shown to have a large repertoire of proteins and enzymes that have been predicted to be strongly involved in the capture and degradation of dietary sugars. In this study we focused on assessing the impact of a specific SusD-like protein on the utilization of dietary sugars. Biochemical evidence, as well as preliminary structural data provides support that this carbohydrate binding protein is capable of binding various dietary sugars. These interactions enable this bacterium to capture sugars derived from the colon and provide an available substrate for membrane bound glycoside hydrolases. The information gathered in this study can shed light on a part of digestion that is unclear at this point in time and create a connection between diet composition and its affect on a dominant member of the gut microbiota.
Importin alpha and nonclassical nuclear localization signal
Authors: Chang Chiung-Wen , Couñago Rafael , Williams Simon , Bodén Mikael , Kobe Boštjan
Published: November 2015

In the classical nuclear import pathway, the specific recognition between the nuclear receptor (importin-α) and the nuclear localization signals (NLSs) plays an essential role on facilitating the cargo import process. Importin-α has two separate NLS-binding sites (the major and the minor sites), accommodate NLSs, comprising of one (monopartite) or two clusters (bipartite) of basic residues connected by a 10 - 12 residue linker. The major NLS-binding site is the preferential binding site for most of the monopartite NLSs characterized to date. By screening random peptide libraries using importin-α variants as bait, the bound NLS sequences could be divided into six classes [1]. The class-3 minor site-specific NLSs and class-5 plant-specific NLSs feature a shorter basic cluster. The molecular basis of the specific binding between these non-classical NLSs and importin-α was not known and in particular, there was a lack of crystal structures of plant importin-α. Here, we present the first crystal structure of plant importin-α, and explain the differential binding specificity between the class-5 plant-specific NLSs and importin-α variants [2]. The binding conformation of the class-3 minor site-specific NLSs features an α-helical turn, that is distinct from the other NLSs reported structurally [3]. Comparative bioinformatic screens not only indicate both plant-specific and minor site-specific NLSs are much less prevalent than the classical NLSs, but also reveal a greater prevalence of these two classes of non-classical NLSs in rice the proteome, compared to the others from yeast, mammals, and even other plant species. Together, our data can help to characterize novel proteins containing non-classical NLSs destined for the cell nucleus by the classical nuclear import pathway.
Structure-Function Relationships of the Collagen-Specific Chaperone Hsp47
Authors: Baumann Ulrich , Gebauer Jan , Widmer Christine , Oecal Sinan , Zaucke Frank
Published: November 2015

Hsp47 is an essential collagen specific chaperone that is crucial for proper formation of the collagen triple helix. KO-mice die at an early emybronic state and missense mutations are linked to severe forms of osteogenesis imperfecta (Ishida & Nagata, 2011). On the other hand, Hsp47 is strongly upregulated in fibrotic pathogenies and has in proof-of-principle studies been successfully targeted . It is a very unusual chaperone as it specifically recognises the folded conformation and releases ist client upon changes in pH and not in an ATP-cycle dependent manner. We have deterined the crystal structure of Hsp47 alone and in complex with homotrimeric collagen model peptides (Widmer et al., 2012), revealing a 2:1 stoichiometry Hsp47:collagen-trimer. The specific recognition of an arginine at the Y-position of the triple helix is explained by a saltbridge to an aspartic acid of Hsp47. Based on these structures we have undergone further investigation in order to shed light on the pH-triggered client release and to gain further insight into client recognition. We have undergone a systematic site-directed mutagenesis study of histidine residues in the binding interface. Interestingly, only few side-chains are predicted to change significantly their protonation state by a pH change from 7 to 6. We have furthermore investigated the influence of the reactive center loop, which is disordered in most crystal structures. An open question is how many binding sites for Hsp47 exist in procollagen. EM studies reveal a large number of specific binding sites, explaining how Hsp47 clamps and stabilises the triple helix and prevents lateral aggregation.
Crystal structure of N-terminal IFIT3 reveals domain-swapping dimerization
Authors: Abbas Yazan , Xie Irene Yuning , Nagar Bhushan
Published: November 2015

IFIT proteins are interferon-inducible, antiviral effectors that form a multiprotein complex with the ability to recognize markers of viral infection and subsequently restrict viruses. IFIT1, IFIT2 and IFIT3 are at the heart of the complex, interacting with each other and several host factors, forming what is known as the 'IFIT interactome'. Central to their ability to mediate complex formation is the tetratricopeptide repeat (TPR) motif, a general protein-protein interaction module comprising a pair of antiparallel alpha helices. Additionally The TPR motifs of IFIT proteins have the unique ability to recognize RNA. Whereas IFIT1 interacts with virus derived ssRNA, IFIT2 has been shown to interact with dsRNA; IFIT3 is not known to bind RNA. Importantly, structural information is available for the N-terminal domain of IFIT1 and full-length IFIT2, but not for IFIT3. To gain insight into the mechanisms regulating complex formation, we are targeting the structure of human IFIT3 before incorporation into the IFIT complex. To that end, we have determined a low resolution crystal structure of N-terminal IFIT3, which reveals a domain swapped dimer. Notably, IFIT3 dimerization is similar to IFIT2, but distinct from IFIT1, which dimerizes via its C-terminus. Sequence conservation and structural analysis suggest that IFIT2 and IFIT3 evolved a similar mechanism for domain swapping. We propose that IFIT2 and IFIT3 may interact by forming domain swapped heterodimers. Current work is aimed at investigating the mechanisms of domain swapping via mutational analysis, and determining the structure of C-terminal human IFIT3.
Predicting Porous Molecular Crystals and Clathrates
Authors: Nyman Jonas , Day Graeme
Published: November 2015

The last decade has seen dramatic improvements in the theories and computer algorithms underlying computational Crystal Structure Predictions [1]. It is now possible to reliably obtain the most likely crystal structures of at least simple molecules starting from nothing more than a drawing of the molecule. We can now go even further and look for rare and exotic kinds of crystals such as porous molecular crystals, clathrates and inclusion compounds among our predictions and calculate their physical properties [2], paving the way for the "science of hypothetical materials". In our poster, we present results on the prediction of fluorophenol xenon clathrates. We have performed crystal structure predictions by global lattice energy searches on o- and m-fluorophenol. The predicted structures have then been analyzed for porosity and their likelihood of being clathrates. From the several thousands of predicted structures, we select a few likely candidates according to an empirical rule based on the guest to host volume ratio [3]. Results from solid state xenon-129 NMR indicate that we have successfully determined the crystal structures of both o- and m-fluorophenol xenon clathrates and we suggest that Crystal Structure Prediction in combination with xenon-129 NMR is a powerful method for determining the structures of clathrates in general.
Synthesis and crystallographic characterization of FAU-NaX Zeolite
Authors: Aparicio Andrea , Quintana Jose , Henao José
Published: November 2015

Zeolites are aluminosilicates with many applications such as ionic exchangers used for the removal of heavy metals (Cd2+) and NH4+ presents in residual water and in farm soil, in air purification and separation of gases like SO2, NO and CO2, in heterogeneous catalysis and as a catalytic support. In the following paper the NaX zeolite was hydrothermally synthetized in an alkaline environment from silicate extracted from rice chaff ashes and post-compsumption aluminum solubilized with NaOH 3M, which were mixed with a relation Si/Al of 2.4 in the reaction crude, then a maturation was made at 250C for 24 hours and 16 hours of aging at 900C. The obtained product was characterized by X-Ray Powder Diffraction Samples, Magnetic Nuclear Resonance of 29Si , Scanning Electron Microscopy, Thermogravimetric Analysis, Differential Scanning Calorimetry and X-Ray Fluorescence (Figure 1). The analysis evidenced the presence of only one crystalline phase corresponding to the NaX zeolite with a relation Si/Al of 1.25. The calculation of the diffraction maxima was performed with Powder X program. The indexing of the diffraction pattern was done using DIVCOL06 program for the twenty (20) first reflections. The determination of the space group was made using the CHEKCELL program. Finally, with all of the reflections was made the refinement of the unit cell constants using the NBS*AIDS83 program. As a result, it was obtained that the NaX crystallized zeolitic phase in a orthorhombic system with a space group Pbcm (No. 57) and cell parameters a=24,975(5) Å, b=17,642(3), c=4,414(8) Å, V=1945,29(2) Å3 y Z=1, with figure of merit M20=21,8 y F30= 34,6 (0,0108, 80).
Desconstruction of Two compounds of p-sulfobenzoic ligand by Reticular Chemistry
Authors: Franco Chris Hebert , Aglio Renata , Corrêa Charlane , Diniz Renata
Published: November 2015

The p-sulfobenzoic (4-psb) acid may be used as precursors for different Metal Organic frameworks (MOFs) since they can display different coordination site on the metal with two different functional groups (carboxylate and sulfonate). Details of the crystal packing can be studied using the Reticular Chemistry like a power tool for the deconstruction process of crystal structure of this compounds formed [1]. Two crystal structures Zn-psb and Mn-psb with 4-psb ligand and Zn+2 and Mn+2 ions, respectively, were synthesized. Single crystal-data were collected using an Oxford GEMINI A-Ultra diffractometer with MoKα radiation (λ = 0.71073 Å) at room temperature (298 K). These structures were refined by SHELXL-97 program [2]. Both compounds crystallized in the triclinic system and space group P-1. Zn-psb structure shows a coordinated Zn atom with a slightly distorted octahedral geometry formed by four oxygen atoms from water molecules with averaged distances at 2.08 (2) Å and two oxygen atoms with distances at 2.12 (2) Å derived from the ligand. Mn-psb structure has coordinated Mn atom with a slightly distorted octahedral geometry formed by four oxygen atoms from sulfonate group coordinated with averaged distances at 2.19 (3) Å and two oxygen atoms from water molecules with distances at 2.17 (2) Å. Despite the structural similarities of the structures, a simple modification of the metal in the reaction leads to a tendency for different network. Thus, Zn-psb structure consists of a network-connected system binodal (3,8). This network is deposited in Reticular Chemistry Structural Resource as a network type tfz-d system with tilling of transitivity [2222] and signature 3[4^2.6^2] + 2[6^3]. However, the Mn-psb structure leads to formation of a regular network with pcu topologic type presenting tilling with transitivity [1111] and signature [4^6]. The system of cavities formed into networks are blocked by ligands in the crystal structure.
Crystallographic data mining - A path to structure prediction in intermetallics?
Authors: Dshemuchadse Julia , Steurer Walter
Published: November 2015

"Many metals adapt very simple structure types, mainly sphere packings such as cubic and hexagonal close-packed, as well as simple body-centered cubic structures. In contrast, around 2 % of all intermetallic crystal structures exhibit unit cells containing 100 or more atoms [1]. These compounds are also termed ""complex intermetallics"" and, while the simplest packings approximate metal atoms as spheres and in doing so explain a number of frequent structure types, the existence of such intricate geometries is not immediately evident. From the analysis of a large number of intermetallic structures, we can recognize geometrical patterns (e.g., recurring building blocks) in order to find out more about their general building principles. Among complex intermetallics, two very distinct phenomena can be observed when inspecting the different structures. On one hand, chemically very diverse compounds can crystallize in rather similar structures, as was shown for a group of more than 40 complex face-centered cubic compounds [2]. On the other hand, small compositional changes within select intermetallic systems can yield a number of different structures, which can also belong to the class of complex intermetallics. A showcase system is found among Al-Cu-Ta, where a small range of the ternary phase diagram contains at least four different structures, all of them complex [3]. By gaining deeper knowledge of the factors influencing structure stabilization, we aim at a better understanding of the mechanisms that are responsible for the formation of intermetallic structures. Thus, we hope to contribute to the quest for explaining why certain intermetallic structures form, while countless other theoretically possible structures do not. Consequently, the prediction of intermetallic structures, especially in still largely unknown parts of multinary phase diagrams, will come into reach."
A total reflection high-energy positron diffraction station at the KEK-SPF
Authors: Wada Ken , Maekawa Masaki , Fukaya Yuki , Mochizuki Izumi , Hyodo Toshio , Shidara Tetsuo , Kawasuso Atsuo
Published: November 2015

A high-intensity mono-energetic positron beam generated by using a linear electron accelerator (linac) provides total reflection high-energy positron diffraction (TRHEPD) researches at Slow Positron Facility (SPF), KEK [1,2]. A pulsed 50-Hz electron beam generated with a dedicated linac (operated at 55 MeV, 0.6 kW) is injected on a Ta converter and causes fast positron-electron pair creation through bremsstrahlung. The positrons showering down on 25 μm-thick W foils, are moderated to thermal energy, and a fraction spontaneously comes out of the foils with an energy of 3 eV owing to the negative work function. The positron converter/moderator assembly is held at an electrostatic voltage of 15 kV for TRHEPD experiment. The emitted positrons are consequently accelerated to 15 keV as they enter the grounded beam-line and are guided by the magnetic field of about 0.015 T to the TRHEPD station. For diffraction experiments, positrons transported by the magnetic field have to be first released into a nonmagnetic region. Since the released positron beam has a large diameter, a brightness-enhancement unit is effectively used to achieve a small-diameter and highly-parallel beam with a sufficient flux. The linac-based positron beam gives about 60 times intensified diffraction pattern from a Si(111)-7x7 reconstructed surface compared to a previous result with a Na-22-based positron beam [3]. An improved signal-to-noise ratio in the obtained pattern due to the intensified beam allowed an observation of clear fractional-order spots in the higher Laue-zones, which had not been observed previously. The much intensified beam with the present system allows adjustment of the sample orientation without accumulating the positron signals. With the brightness enhanced beam, several remarkable results have been obtained efficiently by users of this facility. (Everybody is invited to use KEK Slow Positron Facility through approval of his/her research proposal.)
Crystallographic Aspects of the CDW Instabilities in NbSe3
Authors: Prodan Albert , Van Midden Herman , Zupanič Erik , Žitko Rok , Kusz Joachim , Zubko Maciej , Bennett J.
Published: November 2015

Charge-density waves (CDW) in some quasi one-dimensional compounds can be depinned from the lattice by an external electric field. In the case of NbSe3, two CDW transitions have been reported with onset temperatures of 144 K and 59 K. From an analysis of the published low-temperature (LT) scanning tunneling microscopy (STM) images, which inherently allow the resolution of domain structures on the atomic scale, an alternative model of the CDW modulated structures in NbSe3 is proposed. In contrast to the existing model, where two incommensurate (IC) modes, q1 = (0,0.241,0) and q2 = (0.5,0.260,0.5) are selectively confined to two of three available structurally distinguished types of bi-capped trigonal prismatic columns, the alternative model [1,2] proceeds from the assumption that both columns of the same pair are alternatively modulated by the two modes, whose IC components add within experimental error into a commensurate value. The observed domains are formed as a result of the different bonding within and between the structural layers, separated by Van der Waals gaps, and of the ability of the two modes to be easily interchanged between two symmetry-related columns of the same type. This assumption is in accord with the published LT STM results, which confirm i.a. the presence of both IC modes above 59 K, where according to the previous model only the q1 contribution should be expected. A pair of two alternatively modulated columns of the same type represents the basic structural unit of the CDW ground state. The two modes can formally be replaced by a single inharmonic modulation, obtained by "beating" between the two IC modes.
Visualizing ATP hydrolysis in a viral DNA-packaging molecular motor
Authors: Tang Liang , Zhao Haiyan , Christensen Theodore , Lin Zihan , Lynn Annie
Published: November 2015

Many DNA viruses encode powerful molecular machines to package viral genome into preformed protein shells. These DNA-packaging motors contain an ATPase module that converts the chemical reaction of ATP hydrolysis to physical motion of DNA. We previously determined the structures of the DNA-packaging motor gp2 of Shigella phage Sf6 in the apo form and in complex with ADP and ATP-gamma-S (Zhao et al, 2013, PNAS, 110, 8075). Here we report the structure of gp2 in complex with its substrate ATP at 2.0 Angstrom resolution. To our knowledge, this is the first time to capture, at high resolution, a precatalytic state for ASCE-superfamily ATPases, which include a large group of nucleic acid helicases and translocases involved in a broad range of cellular and viral processes. The structure reveals the precise architecture of the ATP-bound state of the motor immediately prior to catalysis. Comparison with structures of the apo and ADP-complexed forms unveils motions of the Walker A motif coupled with ATP and Mg2+ binding and ATP hydrolysis. In the Walker B motif, residue E118 undergoes a side chain conformational switching coupled with the ATP hydrolysis, whereas residue E119 locks residue R51 side chain to a conformation that is readily reachable to residue E118 side chain. Residue E121 in the Walker B motif deprotonates a water molecule, which acts as a nucleophile to attack the gamma-phosphorous, leading to ATP hydrolysis. The alpha-helix (residue G182-R194) in the linker domain undergoes a translational motion against the ATPase domain triggered by ATP hydrolysis, serving as a mechanism for translating the energy from the chemical reaction into physical movement of DNA. We further observed the time course of ATP hydrolysis by gp2 by determining structures of gp2:ATP complexes captured at various incubation time. These structures have made it possible to delineate, at atomic detail, the complete cycle of ATP hydrolysis of this viral DNA-packaging molecular motor.
Structure and function of cement proteins in human adenovirus
Authors: Reddy Vijay , Nemerow Glen
Published: November 2015

Human adenoviruses (HAdVs) are large (~150nm in diameter, 150MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause respiratory, ocular, and enteric diseases. The capsid shell of adenovirus (Ad) comprises multiple copies of three major capsid proteins (MCP: hexon, penton base and fiber) and four minor/cement proteins (IIIa, VI, VIII and IX) that are organized with pseudo T=25 icosahedral symmetry. In addition, six other proteins (V, VII, μ, IVa2, terminal protein and protease) are encapsidated along with the 36Kb dsDNA genome inside the capsid. The crystal structures of all three MCPs are known and so is their organization in the capsid from prior X-ray crystallography and cryoEM analyses. However structures and locations of various cement proteins are of considerable debate. We have determined and refined the structure of an entire human adenovirus employing X-ray crystallpgraphic methods at 3.8Å resolution. Adenovirus cement proteins play crucial roles in virion assembly, disassembly, cell entry and infection. Based on the refined crystal structure of adenovirus, we have determined the structure of the cement protein VI, a key membrane-lytic molecule and its associations with proteins V and VIII, which together glue peripentonal hexons beneath vertex region and connect them to rest of the capsid. Following virion maturation, the cleaved N-terminal pro-peptide of VI is observed deep in the peripentonal hexon cavity, detached from the membrane-lytic domain. Furthermore, we have significantly revised the recent cryoEM models for proteins IIIa and IX and both are located on the capsid exterior. Together, the cement proteins exclusively stabilize the hexon shell, thus rendering penton vertices the weakest links of the adenovirus capsid. Adenovirus cement protein structures reveal the molecular basis of the maturation cleavage of VI that is needed for endosome rupture and delivery of the virion into cytoplasm.
Investigation of exponential RNA amplification by the Qβ replicase complex
Authors: Olesen Heidi , Knudsen Charlotte , Seweryn Paulina , Brodersen Ditlev , Kutlubaeva Zarina , Chetverin Alexander , Mulder Frans , Jensen Lars Alf , Yoshimura Yuichi
Published: November 2015

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.
Nidovirus papain-like proteases antagonize the host innate immune response
Authors: Bailey-Elkin Ben , van Kasteren Puck , Snijder Eric , Kikkert Marjolein , Mark Brian
Published: November 2015

Protein ubiquitination regulates important innate immune responses. Ubiquitin (Ub) can be attached to lysine residues on cellular proteins to promote, among other activities, the innate immune responses of the cell. These pathways can in turn be downregulated by the removal of Ub from cellular proteins by deubiquitinases (DUBs). Viruses of the order Nidovirales have positive-sense, single stranded RNA genomes. Within this order are the families Coronaviridae and Arteriviridae, which include viruses known to cause severe disease in humans and animals, respectively. Members of the families Coronaviridae and Arteriviridae share a common mechanism of gene expression, whereby the viral nonstructural proteins (nsps) are initially expressed as a single polyprotein, which is then cleaved into functional units by papain-like protease (PLP) domains encoded within. Interestingly, while also being necessary for viral replication, a number of Nidovirus PLPs have been shown to remove Ub from host proteins, in order to down-regulate the host innate immune response. Here we present the crystal structure of a Nidovirus PLP in complex with Ub. The structure allowed for the characterization of a Ub-binding interface, and identification of specific residues involved in Ub recognition that are distant from the enzyme active site.  The selective inactivation of DUB activity of viral PLP enzymes verses their polyprotein cleavage activity by site directed mutagenesis is allowing us to understand the role of DUB activity in evading innate immune responses of the host, and opens the door for the development of improved live attenuated vaccines against Nidoviruses and other viruses encoding similar dual specificity proteases.