Production, purification and crystallization of a trans-sialidase from Trypanosoma vivax
Sialidases and trans-sialidases play important roles in the life cycles of various microorganisms. These enzymes can serve nutritional purposes, act as virulence factors or mediate cellular interactions (cell evasion and invasion). In the case of the protozoan parasite Trypanosoma vivax, trans-sialidase activity has been suggested to be involved in infection-associated anaemia, which is the major pathology in the disease nagana. The physiological role of trypanosomal trans-sialidases in host–parasite interaction as well as their structures remain obscure. Here, the production, purification and crystallization of a re...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Haynes, C.L.F.Ameloot, P.Remaut, H.Callewaert, N.Sterckx, Y.G.-J.Magez, S. Tags: trans-sialidase Trypanosoma vivax research communications Source Type: research

Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP
Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The Cα backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Lorimer, D.D.Choi, R.Abramov, A.Nakazawa Hewitt, S.Gardberg, A.S.Van Voorhis, W.C.Staker, B.L.Myler, P.J.Edwards, T.E. Tags: hydrolase structural genomics Seattle Structural Genomics Center for Infectious Disease SSGCID metal-binding protein research communications Source Type: research

Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia
The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the ...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Abendroth, J.Choi, R.Wall, A.Clifton, M.C.Lukacs, C.M.Staker, B.L.Van Voorhis, W.Myler, P.Lorimer, D.D.Edwards, T.E. Tags: aspartate aminotransferase structural genomics Seattle Structural Genomics Center for Infectious Disease pyridoxalphosphate lysine Giardia lamblia Trypanosoma brucei Leishmania major research communications Source Type: research

Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it ...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Edwards, T.E.Gardberg, A.S.Phan, I.Q.H.Zhang, Y.Staker, B.L.Myler, P.J.Lorimer, D.D. Tags: SSGCID Entamoeba histolytica amoebic dysentery parasitic protozoan allosteric regulation cell-wall biogenesis GlcNAc research communications Source Type: research

The X-ray structure of Plasmodium falciparum dihydroorotate dehydrogenase bound to a potent and selective N-phenylbenzamide inhibitor reveals novel binding-site interactions
Plasmodium species are protozoan parasites that are the causative agent of malaria. Malaria is a devastating disease, and its treatment and control have been hampered by the propensity of the parasite to become drug-resistant. Dihydroorotate dehydrogenase (DHODH) has been identified as a promising new target for the development of antimalarial agents. Here, the X-ray structure of P. falciparum DHODH bound to a potent and selective N-phenylbenzamide-based inhibitor (DSM59) is described at 2.3 Å resolution. The structure elucidates novel binding-site interactions and shows how conformational flexibility of the enzyme...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Deng, X.Matthews, D.Rathod, P.K.Phillips, M.A. Tags: dihydroorotate dehydrogenase Plasmodium falciparum DSM59 research communications Source Type: research

Recombinant production, crystallization and crystal structure determination of dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis
In this study, a reproducible protocol for the heterologous expression of active dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis (LbDHODH) was developed and its crystal structure was determined at 2.12 Å resolution. L. (V.) braziliensis is the species responsible for the mucosal form of leishmaniasis, a neglected disease for which no cure or effective therapy is available. Analyses of sequence, structural and kinetic features classify LbDHODH as a member of the class 1A DHODHs and reveal a very high degree of structural conservation with the previously reported structures of orthologous trypanos...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Reis, R.A.G.Lorenzato, E.Silva, V.C.Nonato, M.C. Tags: dihydroorotate dehydrogenase Leishmania (Viannia) braziliensis leishmaniasis research communications Source Type: research

The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly
This study provides a reagent and template to support further work in this area. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Barrack, K.L.Fyfe, P.K.Hunter, W.N. Tags: chaperone helical fold protein – protein interactions selenomethionine tubulin-binding protein research communications Source Type: research

Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity
Inosine 5′-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH (CpIMPDH) in complex with inosine 5′-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compound...
Source: Acta Crystallographica Section F - April 21, 2015 Category: Biochemistry Authors: Kim, Y.Makowska-Grzyska, M.Gorla, S.K.Gollapalli, D.R.Cuny, G.D.Joachimiak, A.Hedstrom, L. Tags: Cryptosporidium inosine 5 ′ -monophosphate dehydrogenase P131 research communications Source Type: research

Structure of a CutA1 divalent-cation tolerance protein from Cryptosporidium parvum, the protozoal parasite responsible for cryptosporidiosis
Cryptosporidiosis is an infectious disease caused by protozoan parasites of the Cryptosporidium genus. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of C. parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, which is a toxic element in excess. Here, the crystal structure of this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution. As observed for other CutA1 structures, the 117-residu...
Source: Acta Crystallographica Section F - April 18, 2015 Category: Biochemistry Authors: Buchko, G.W.Abendroth, J.Clifton, M.C.Robinson, H.Zhang, Y.Hewitt, S.N.Staker, B.L.Edwards, T.E.Van Voorhis, W.C.Myler, P.J. Tags: Cryptosporidium parvum divalent-cation tolerance protein CutA1 cryptosporidiosis research communications Source Type: research

Solution-state NMR structure of the putative morphogene protein BolA (PFE0790c) from Plasmodium falciparum
Protozoa of the genus Plasmodium are responsible for malaria, which is perhaps the most important parasitic disease to infect mankind. The emergence of Plasmodium strains resistant to current therapeutics and prophylactics makes the development of new treatment strategies urgent. Among the potential targets for new antimalarial drugs is the BolA-like protein PFE0790c from Plasmodium falciparum (Pf-BolA). While the function of BolA is unknown, it has been linked to cell morphology by regulating transcription in response to stress. Using an NMR-based method, an ensemble of 20 structures of Pf-BolA was determined and deposite...
Source: Acta Crystallographica Section F - April 18, 2015 Category: Biochemistry Authors: Buchko, G.W.Yee, A.Semesi, A.Myler, P.J.Arrowsmith, C.H.Hui, R. Tags: Plasmodium falciparum BolA PFE0790c research communications Source Type: research

Towards a molecular understanding of the apicomplexan actin motor: on a road to novel targets for malaria remedies?
Apicomplexan parasites are the causative agents of notorious human and animal diseases that give rise to considerable human suffering and economic losses worldwide. The most prominent parasites of this phylum are the malaria-causing Plasmodium species, which are widespread in tropical and subtropical regions, and Toxoplasma gondii, which infects one third of the world's population. These parasites share a common form of gliding motility which relies on an actin–myosin motor. The components of this motor and the actin-regulatory proteins in Apicomplexa have unique features compared with all other eukaryotes. This, tog...
Source: Acta Crystallographica Section F - April 16, 2015 Category: Biochemistry Authors: Kumpula, E.-P.Kursula, I. Tags: malaria actin polymerization cytoskeleton regulation gliding motility drug design parasitology research communications Source Type: research

Three-dimensional structures in the design of therapeutics targeting parasitic protozoa: reflections on the past, present and future
Parasitic protozoa cause a range of diseases which threaten billions of human beings. They are responsible for tremendous mortality and morbidity in the least-developed areas of the world. Presented here is an overview of the evolution over the last three to four decades of structure-guided design of inhibitors, leads and drug candidates aiming at targets from parasitic protozoa. Target selection is a crucial and multi-faceted aspect of structure-guided drug design. The major impact of advances in molecular biology, genome sequencing and high-throughput screening is touched upon. The most advanced crystallographic techniqu...
Source: Acta Crystallographica Section F - April 16, 2015 Category: Biochemistry Authors: Hol, W.G.J. Tags: parasitic protozoa antiparasitic drug discovery research communications Source Type: research

Large-volume protein crystal growth for neutron macromolecular crystallography
Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large c...
Source: Acta Crystallographica Section F - March 30, 2015 Category: Biochemistry Authors: Ng, J.D.Baird, J.K.Coates, L.Garcia-Ruiz, J.M.Hodge, T.A.Huang, S. Tags: large-volume crystals neutron macromolecular crystallography research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus
In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.8, b = 86.0, c = 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-re...
Source: Acta Crystallographica Section F - March 27, 2015 Category: Biochemistry Authors: Rosário, A.L.Sena, F.V.Batista, A.P.Oliveira, T.F.Athayde, D.Pereira, M.M.Brito, J.A.Archer, M. Tags: NDH-II NADH dehydrogenase respiratory chain Staphylococcus aureus research communications Source Type: research

Serendipitous crystallization and structure determination of cyanase (CynS) from Serratia proteamaculans
Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS from Serratia proteamaculans (SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space group P1 and diffracted to 2.1 Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS from Escherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Ad...
Source: Acta Crystallographica Section F - March 21, 2015 Category: Biochemistry Authors: Butryn, A.Stoehr, G.Linke-Winnebeck, C.Hopfner, K.-P. Tags: CynS cyanase cyanate hydratase research communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis
This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa with a C-terminal His6 tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space group P4322, with unit-cell parameters a = b = 101.581, c = 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1 and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress. (Sour...
Source: Acta Crystallographica Section F - March 21, 2015 Category: Biochemistry Authors: Ding, R.Xu, C.Chen, X.Bao, M.Qiu, X. Tags: Microcystis aeruginosa aeruginosin 2-carboxy-6-hydroxyoctahydroindole moiety AerF research communications Source Type: research

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod – Zwilch – ZW10 (RZZ) complex
The spindle-assembly checkpoint (SAC) monitors kinetochore – microtubule attachment during mitosis. In metazoans, the three-subunit Rod – Zwilch – ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1 – Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼ 800   kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ comp...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Altenfeld, A. Wohlgemuth, S. Wehenkel, A. Vetter, I.R. Musacchio, A. Tags: spindle-assembly checkpoint cell division mitosis RZZ complex Rod Zwilch ZW10 kinetochore Ndc80 Mis12 Knl1 KMN network research communications Source Type: research

Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 β -N-acetylglucosaminidase from the marine bacterium Vibrio harveyi
In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1   M sodium acetate pH 4.6, 1.4   M sodium malonate, while crystals of the D437A mutant were obtained in 0.1   M bis-tris pH 7.5, 0.1   M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5   Å . All crystals were composed of the same...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Meekrathok, P. B ü rger, M. Porfetye, A.T. Vetter, I.R. Suginta, W. Tags: β -N-acetylglucosaminidase Vibrio harveyi GH20 glycoside hydrolase family research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of an endo-1,4- β -d-glucanase from Aspergillus aculeatus F-50
Cellulose is the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in a wide variety of industries. Endo-1,4- β -d-glucanase (EC 3.2.1.4; endoglucanase), which can catalyze the random hydrolysis of β -1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase) that was classified into glycoside hydrolase family 12 has been found to be effectively expressed in the industrial strain Pichia pastoris. Here, recombinant FI-CMCase was crystallized. Crystals belonging to the...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Chen, Y. Huang, J.-W. Chen, C.-C. Lai, H.-L. Jin, J. Guo, R.-T. Tags: cellulase endoglucanase Aspergillus industrial enzyme research communications Source Type: research

Crystallization and preliminary X-ray analysis of four cysteine proteases from Ficus carica latex
The latex of the common fig (Ficus carica) contains a mixture of at least five cysteine proteases commonly known as ficins (EC 3.4.22.3). Four of these proteases were purified to homogeneity and crystals were obtained in a variety of conditions. The four ficin (iso)forms appear in ten different crystal forms. All diffracted to better than 2.10 Å resolution and for each form at least one crystal form diffracted to 1.60 Å resolution or higher. Ficin (iso)forms B and C share a common crystal form, suggesting close sequence and structural similarity. The latter diffracted to a resolution of 1.20 Å and b...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Haesaerts, S.Rodriguez Buitrago, J.A.Loris, R.Baeyens-Volant, D.Azarkan, M. Tags: ficin cysteine protease papain Ficus carica research communications Source Type: research

Analytical ultracentrifugation and preliminary X-ray studies of the chloroplast envelope quinone oxidoreductase homologue from Arabidopsis thaliana
Quinone oxidoreductases reduce a broad range of quinones and are widely distributed among living organisms. The chloroplast envelope quinone oxidoreductase homologue (ceQORH) from Arabidopsis thaliana binds NADPH, lacks a classical N-terminal and cleavable chloroplast transit peptide, and is transported through the chloroplast envelope membrane by an unknown alternative pathway without cleavage of its internal chloroplast targeting sequence. To unravel the fold of this targeting sequence and its substrate specificity, ceQORH from A. thaliana was overexpressed in Escherichia coli, purified and crystallized. Crystals of apo ...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Mas y mas, S.Giustini, C.Ferrer, J.-L.Rolland, N.Curien, G.Cobessi, D. Tags: chloroplast envelope quinone oxidoreductase homologue analytical ultracentrifugation research communications Source Type: research

Structure of dihydrodipicolinate synthase from the commensal bacterium Bacteroides thetaiotaomicron at 2.1 Å resolution
Dihydrodipicolinate synthase (DapA) catalyzes the first committed step of the diaminopimelate biosynthetic pathway of lysine. It has been shown to be an essential enzyme in many bacteria and has been the subject of research to generate novel antibiotics. However, this pathway is present in both pathogenic and commensal bacteria, and antibiotics targeting DapA may interfere with normal gut colonization. Bacteroides thetaiotaomicron is a Gram-negative commensal bacterium that makes up a large proportion of the normal microbiota of the human gut. The structure of DapA from B. thetaiotaomicron (BtDapA) has been determined. Thi...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Mank, N.Arnette, A.Klapper, V.Offermann, L.Chruszcz, M. Tags: dihydrodipicolinate synthase Bacteroides thetaiotaomicron research communications Source Type: research

Structure of the ABL2/ARG kinase in complex with dasatinib
ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) and ABL1 genes. Similarly, fusion of the ETV6 (Tel) and ARG genes drives some forms of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine ki...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Ha, B.H.Simpson, M.A.Koleske, A.J.Boggon, T.J. Tags: ABL2 ARG research communications Source Type: research

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex
The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was pur...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Altenfeld, A.Wohlgemuth, S.Wehenkel, A.Vetter, I.R.Musacchio, A. Tags: spindle-assembly checkpoint cell division mitosis RZZ complex Rod Zwilch ZW10 kinetochore Ndc80 Mis12 Knl1 KMN network research communications Source Type: research

Purification, crystallization and preliminary X-ray crystallographic analysis of the phosphatase domain (PA3346PD) of the response regulator PA3346 from Pseudomonas aeruginosa PAO1
The phosphatase domain (PA3346PD) of the response regulator PA3346 modulates the downstream anti-anti-σ factor PA3347 to regulate swarming motility in Pseudomonas aeruginosa PAO1. PA3346PD, which comprises the protein phosphatase 2C domain (PP2C), is classified as a Ser/Thr phosphatase of the Mg2+- or Mn2+-dependent protein phosphatase (PPM) family. The recombinant PA3346PD, with molecular mass 26 kDa, was overexpressed in Escherichia coli, purified on an Ni2+–NTA agarose column and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected from PA3346PD crystals to a reso...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Chen, L.-Y.Wu, P.-H.Guan, H.-H.Fun, H.-K.Chang, H.-Y.Chen, C.-J. Tags: phosphatase domain response regulator Pseudomonas aeruginosa research communications Source Type: research

Expression, purification, crystallization and preliminary crystallographic analysis of a GH20 β-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi
In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å. All crystals were composed of the same type of di...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Meekrathok, P.Bürger, M.Porfetye, A.T.Vetter, I.R.Suginta, W. Tags: β -N-acetylglucosaminidase Vibrio harveyi GH20 glycoside hydrolase family research communications Source Type: research

Structure of the omalizumab Fab
Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalia...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Jensen, R.K.Plum, M.Tjerrild, L.Jakob, T.Spillner, E.Andersen, G.R. Tags: IgE Fab fragment therapeutic antibody research communications Source Type: research

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1–Ter DNA complex
This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Jaiswal, R.Singh, S.K.Bastia, D.Escalante, C.R. Tags: protein – DNA complex Reb1 transcription termination eukaryotic RNA Pol I research communications Source Type: research

Cloning, expression, crystallization and preliminary X-ray studies of a superfolder GFP fusion of cyanobacterial Psb32
A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P6122. A full data set extending to 2.3 Å resolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP and AtTLP18.3 from Arabidopsis thaliana as search models. Based on this expression construct, a versatile library of 24 vectors combining four different supe...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Liauw, P.Kannchen, D.Gasper, R.Dyczmons-Nowaczyk, N.Nowaczyk, M.M.Hofmann, E. Tags: Psb32 superfolder GFP Thermosynechococcus elongatus BP-1 research communications Source Type: research

Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase from Eupenicillium terrenum
This study reveals the first structural details of group I FPODs that prefer α-fructosyl substrates and could provide significant useful information for uncovering the mechanism of substrate specificity of FAOD/FPODs and guidance towards future enzyme engineering for diagnostic purposes. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Gan, W.Gao, F.Xing, K.Jia, M.Liu, H.Gong, W. Tags: fructosyl peptide oxidase Eupenicillium terrenum FAOD/FPOD family research communications Source Type: research

Crystallization of mutants of Turnip yellow mosaic virus protease/ubiquitin hydrolase designed to prevent protease self-recognition
Processing of the polyprotein of Turnip yellow mosaic virus is mediated by the protease PRO. PRO cleaves at two places, one of which is at the C-terminus of the PRO domain of another polyprotein molecule. In addition to this processing activity, PRO possesses an ubiquitin hydrolase (DUB) activity. The crystal structure of PRO has previously been reported in its polyprotein-processing mode with the C-terminus of one PRO inserted into the catalytic site of the next PRO, generating PRO polymers in the crystal packing of the trigonal space group. Here, two mutants designed to disrupt specific PRO–PRO interactions were ge...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Ayach, M.Bressanelli, S. Tags: self-processing viral cysteine proteinase ubiquitin hydrolase research communications Source Type: research

The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway
Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a ...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Dalle Vedove, A.Lucarelli, A.P.Nardone, V.Matino, A.Parisini, E. Tags: cadherin dimerization mechanism cell adhesion research communications Source Type: research

Purification, crystallization and preliminary X-ray analysis of the periplasmic haem-binding protein HutB from Vibrio cholerae
The mechanism of haem transport across the inner membrane of pathogenic bacteria is currently insufficiently understood at the molecular level and no information is available for this process in Vibrio cholerae. To obtain structural insights into the periplasmic haem-binding protein HutB from V. cholerae (VcHutB), which is involved in haem transport through the HutBCD haem-transport system, at the atomic level, VcHutB was cloned, overexpressed and crystallized using 1.6 M ammonium sulfate as a precipitant at pH 7.0. X-ray diffraction data were collected to 2.4 Å resolution on the RRCAT PX-BL-21 beamline at the In...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Agarwal, S.Biswas, M.Dasgupta, J. Tags: Vibrio cholerae iron uptake periplasmic haem-binding protein ABC transporter research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of an endo-1,4-β-d-glucanase from Aspergillus aculeatus F-50
Cellulose is the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in a wide variety of industries. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which can catalyze the random hydrolysis of β-1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase) that was classified into glycoside hydrolase family 12 has been found to be effectively expressed in the industrial strain Pichia pastoris. Here, recombinant FI-CMCase was crystallized. Crystals belonging to the or...
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Chen, Y.Huang, J.-W.Chen, C.-C.Lai, H.-L.Jin, J.Guo, R.-T. Tags: cellulase endoglucanase Aspergillus industrial enzyme research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the interaction of Aeromonas hydrophila MtaN-1 with S-adenosylhomocysteine
In this study, MtaN-1 from Aeromonas hydrophila was successfully expressed and purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of the protein in complex with the substrate SAH were obtained and diffracted to a resolution of 1.4 Å. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 102.7, c = 118.8 Å. The asymmetric unit contained two molecules of MtaN-1 complexed with SAH. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - March 20, 2015 Category: Biochemistry Authors: Xu, Y.Quan, C.-S.Jin, X.Jin, X.Zhao, J.Jin, L.Kim, J.-S.Guo, J.Fan, S.Ha, N.-C. Tags: Aeromonas hydrophila MtaN-1 S-adenosylmethionine research communications Source Type: research

Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097
The secreted cystine-knot protein sclerostin was first identified from genetic screening of patients suffering from the rare bone-overgrowth diseases sclerosteosis and van Buchem disease. Sclerostin acts a negative regulator of bone growth through inhibiting the canonical Wnt signalling cascade by binding to and blocking the Wnt co-receptor LRP5/6. Its function in blocking osteoblastogenesis makes it an important target for osteoanabolic therapy approaches to treat osteoporosis, which is characterized by a progressive decrease in bone mass and density. In this work, the production, crystallization and preliminary X-ray dif...
Source: Acta Crystallographica Section F - March 19, 2015 Category: Biochemistry Authors: Boschert, V.Muth, E.-M.Knappik, A.Frisch, C.Mueller, T.D. Tags: Wnt signalling pathway sclerostin bone homeostasis osteoporosis research communications Source Type: research

Crystallization and preliminary X-ray analysis of Rv1674c from Mycobacterium tuberculosis
Adaptations to hypoxia play an important role in Mycobacterium tuberculosis pathogenesis. Rv0324, which contains an HTH DNA-binding domain and a rhodanese domain, is one of the key transcription regulators in response to hypoxia. M. tuberculosis Rv1674c is a homologue of Rv0324. To understand the interdomain interaction and regulation of the HTH domain and the rhodanese domain, recombinant Rv1674c protein was purified and crystallized by the vapour-diffusion method. The crystals diffracted to 2.25 Å resolution. Preliminary diffraction analysis suggests that the crystals belonged to space group P3121 or P3221, with ...
Source: Acta Crystallographica Section F - February 28, 2015 Category: Biochemistry Authors: Li, J.Wang, X.Gong, W.Niu, C.Zhang, M. Tags: Rv1674c HTH DNA-binding domain rhodanese domain research communications Source Type: research

Origin and use of crystallization phase diagrams
Crystallization phase diagrams are frequently used to conceptualize the phase relations and also the processes taking place during the crystallization of macromolecules. While a great deal of freedom is given in crystallization phase diagrams owing to a lack of specific knowledge about the actual phase boundaries and phase equilibria, crucial fundamental features of phase diagrams can be derived from thermodynamic first principles. Consequently, there are limits to what can be reasonably displayed in a phase diagram, and imagination may start to conflict with thermodynamic realities. Here, the commonly used `crystallizatio...
Source: Acta Crystallographica Section F - February 27, 2015 Category: Biochemistry Authors: Rupp, B. Tags: crystallization phase diagrams non-ideal mixtures thermodynamics excess properties research communications Source Type: research

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1)
The post-transcriptional addition of uridines to the 3′-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3′-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate micr...
Source: Acta Crystallographica Section F - February 21, 2015 Category: Biochemistry Authors: Yates, L.A.Durrant, B.P.Barber, M.Harlos, K.Fleurdépine, S.Norbury, C.J.Gilbert, R.J.C. Tags: terminal uridylyltransferases Cid1 research communications Source Type: research

High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-l-arabinofuranosidase activity
A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Å resolution. As for other GH43 members, the enzyme folds as a five-bladed β-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in com...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Hassan, N.Kori, L.D.Gandini, R.Patel, B.K.C.Divne, C.Tan, T.C. Tags: glycoside hydrolase five-bladed β -propeller arabinofuranosidase Halothermothrix orenii halothermophile research communications Source Type: research

Crystallization and preliminary X-ray analysis of the Plasmodium falciparum apicoplast DNA polymerase
Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystal...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Milton, M.E.Choe, J.Honzatko, R.B.Nelson, S.W. Tags: DNA polymerase apicoplast Plasmodium falciparum research communications Source Type: research

Protein production, crystallization and preliminary X-ray analysis of two isoforms of the Dscam1 Ig7 domain
Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) plays a critical role in neural development. It can potentially form 38 016 isoforms through alternative RNA splicing, and exhibits isoform-specific homophilic interaction through three variable Ig domains (Ig2, Ig3 and Ig7). The diversity and homophilic interaction are essential for its functions. Ig7 has 33 isoforms and is the most variable among the three variable Ig domains. However, only one isoform of Ig7 (isoform 30) has been structurally determined to date. Here, two isoforms of Dscam1 Ig7 (isoforms 5 and 9; Ig75 and Ig79) were produced and crystallized. ...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Li, S.-A.Cheng, L.Yu, Y.Chen, Q. Tags: Dscam1 RNA splicing Ig7 homophilic dimer research communications Source Type: research

Bacterial expression and preliminary crystallographic studies of a 149-residue fragment of human Caprin-1
Caprin-1 is an RNA-binding protein which plays critical roles in several important biological processes, including cellular proliferation, the interferon-mediated antiviral innate immune response, the maintenance of synaptic plasticity and the formation of RNA stress granules. Caprin-1 has been implicated in the pathogenesis of several human diseases, including osteosarcoma, breast cancer, viral infections, hearing loss and neurodegenerative disorders. Despite the emerging biological and physiopathological significance of Caprin-1, no structural information is available for this protein. Moreover, Caprin-1 does not have se...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Wu, Y.Zhu, J.Huang, X.Du, Z. Tags: Caprin-1 Caprin-2 G3BP1 Pou4f3 JEV core protein DENV-2 sfRNA research communications Source Type: research

Crystallization and preliminary crystallographic analysis of acetophenone reductase from Geotrichum candidum NBRC 4597
Acetophenone reductase (APRD) from Geotrichum candidium NBRC 4597 was crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant. The crystal belonged to space group P6522, with unit-cell parameters a = b = 104.5, c = 273.7 Å, and diffracted to 2.6 Å resolution. Phasing using the single-wavelength anomalous diffraction method was successful. Model building and crystallographic refinement are in progress. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Sugiyama, Y.Senda, M.Senda, T.Matsuda, T. Tags: acetophenone reductase enantioselectivity asymmetric reduction research communications Source Type: research

The structure of C290A:C393A Aurora A provides structural insights into kinase regulation
Aurora A is a Ser/Thr protein kinase that functions in cell-cycle regulation and is implicated in cancer development. During mitosis, Aurora A is activated by autophosphorylation on its activation loop at Thr288. The Aurora A catalytic domain (amino acids 122–403) expressed in Escherichia coli autophosphorylates on two activation-loop threonine residues (Thr288 and Thr287), whereas a C290A,C393A double point mutant of the Aurora A catalytic domain autophosphorylates only on Thr288. The structure of the complex of this mutant with ADP and magnesium was determined to 2.1 Å resolution using molecular replacement...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Burgess, S.G.Bayliss, R. Tags: protein kinase phosphorylation activation research communications Source Type: research

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX)
In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Campos, B.M.Liberato, M.V.Polikarpov, I.Zeri, A.C.M.Squina, F.M. Tags: accessory domain carbohydrate-binding module research communications Source Type: research

Mode of binding of the antithyroid drug propylthiouracil to mammalian haem peroxidases
The mammalian haem peroxidase superfamily consists of myeloperoxidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). These enzymes catalyze a number of oxidative reactions of inorganic substrates such as Cl−, Br−, I− and SCN− as well as of various organic aromatic compounds. To date, only structures of MPO and LPO are known. The substrate-binding sites in these enzymes are located on the distal haem side. Propylthiouracil (PTU) is a potent antithyroid drug that acts by inhibiting the function of TPO. It has also been shown to inhibit the action of LPO. Howeve...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Singh, R.P.Singh, A.Kushwaha, G.S.Singh, A.K.Kaur, P.Sharma, S.Singh, T.P. Tags: lactoperoxidase propylthiouracil distal haem side antithyroid drug research communications Source Type: research

Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide
In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components re...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Bradshaw, W.J.Roberts, A.K.Shone, C.C.Acharya, K.R. Tags: Clostridium difficile surface layer Cwp84 host – pathogen interactions research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor ArgR from Bacillus halodurans
The arginine repressor (ArgR) is a transcriptional regulator which regulates genes encoding proteins involved in arginine biosynthesis and the arginine catabolic pathway. ArgR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. ArgR (Bh2777) from B. halodurans is composed of 149 amino-acid residues with a molecular mass of 16 836 Da. ArgR was crystallized at 296 K using 1,2-propanediol as a precipitant. Crystals of N-terminally His-tagged ArgR were obtained by the sitting-drop vapour-diffusion method. Dehydrated crystals showed a dramatic improvement in diffraction qu...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Kang, J.Park, Y.W.Yeo, H.K.Lee, J.Y. Tags: ArgR transcriptional regulator dehydration research communications Source Type: research

Structures of Escherichia coli tryptophanase in holo and `semi-holo' forms
Two crystal forms of Escherichia coli tryptophanase (tryptophan indole-lyase, Trpase) were obtained under the same crystallization conditions. Both forms belonged to the same space group P43212 but had slightly different unit-cell parameters. The holo crystal form, with pyridoxal phosphate (PLP) bound to Lys270 of both polypeptide chains in the asymmetric unit, diffracted to 2.9 Å resolution. The second crystal form diffracted to 3.2 Å resolution. Of the two subunits in the asymmetric unit, one was found in the holo form, while the other appeared to be in the apo form in a wide-open conformation with two su...
Source: Acta Crystallographica Section F - February 19, 2015 Category: Biochemistry Authors: Kogan, A.Raznov, L.Gdalevsky, G.Y.Cohen-Luria, R.Almog, O.Parola, A.H.Goldgur, Y. Tags: tryptophanase Escherichia coli polymorphism research communications Source Type: research