1,721,079 research outputs found
Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human spliceosomal protein U5-15kD
No full textThe gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding, The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 62.3, b = 65.7, c = 37.1 Angstrom. They diffract to at least 3.0 Angstrom and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection
Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation
No full textThe crystal structure of a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44, was solved by means of molecular replacement and refined at 1.84 Angstrom. This is the first structure of a cold-adapted subtilase to be determined and its elucidation facilitates examination of the molecular principles underlying temperature adaptation in enzymes. The cold-adapted Vibrio proteinase was compared with known three-dimensional structures of homologous enzymes of meso- and thermophilic origin, proteinase K and thermitase, to which it has high structural resemblance. The main structural features emerging as plausible determinants of temperature adaptation in the enzymes compared involve the character of their exposed and buried surfaces, which may be related to temperature-dependent variation in the physical properties of water. Thus, the hydrophobic effect is found to play a significant role in the structural stability of the meso- and thermophile enzymes, whereas the cold-adapted enzyme has more of its apolar surface exposed. In addition, the cold-adapted Vibrio proteinase is distinguished from the more stable enzymes by its strong anionic character arising from the high occurrence of uncompensated negatively charged residues at its surface. Interestingly, both the cold-adapted and thermophile proteinases differ from the mesophile enzyme in having more extensive hydrogen- and ion pair interactions in their structures; this supports suggestions of a dual role of electrostatic interactions in the adaptation of enzymes to both high and low temperatures. The Vibrio proteinase has three calcium ions associated with its structure, one of which is in a calcium-binding site not described in other subtilases
Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases
No full textThe Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin. It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active hole-forms by 4'-phosphopantetheinylation. The B. subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized. Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant. The crystals belong to the tetragonal space group P4(1)2(1)2/P4(3)2(1)2, with unit-cell parameters a = b = 65.3, c = 150.5 Angstrom. They diffract beyond 2.8 Angstrom and contain one molecule in the asymmetric unit
Structure of the novel alpha amylase AmyC from Thermotoga maritima
No full textalpha-Amylases are essential enzymes in alpha-glucan metabolism and catalyse the hydrolysis of long sugar polymers such as amylose and starch. The crystal structure of a previously unidentified amylase (AmyC) from the hyperthermophilic organism Thermotoga maritima was determined at 2.2 angstrom resolution by means of MAD. AmyC lacks sequence similarity to canonical alpha-amylases, which belong to glycosyl hydrolase families 13, 70 and 77, but exhibits significant similarity to a group of as yet uncharacterized proteins in COG1543 and is related to glycerol hydrolase family 57 (GH-57). AmyC reveals features that are characteristic of alpha-amylases, such as a distorted TIM-barrel structure formed by seven beta-strands and alpha-helices ( domain A), and two additional but less well conserved domains. The latter are domain B, which contains three helices inserted in the TIM-barrel after beta-sheet 2, and domain C, a five-helix region at the C-terminus. Interestingly, despite moderate sequence homology, structure comparison revealed significant similarities to a member of GH-57 with known three-dimensional structure, Thermococcus litoralis 4-glucanotransferase, and an even higher similarity to a structure of an enzyme of unknown function from Thermus thermophilus
Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4 '-phosphopantetheinyl transferase superfamily
Full text linkThe Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the SfpCoA complex determined at 1.8 Angstrom resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry, This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase, The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate
Screening orthologs as an important variable in crystallization: preliminary X-ray diffraction studies of the tRNA-modifying enzyme S-adenosylmethionine : tRNA ribosyl transferase/isomerase
No full textThe genes encoding the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyl transferase/isomerase (QueA) from 12 eubacterial sources were overexpressed in Escherichia coli and the resulting products were purified to homogeneity and subjected to crystallization trials. Using the hanging-drop vapour-diffusion method, crystals suitable for X-ray diffraction experiments were only obtained for the queA gene product from Bacillus subtilis. The crystals belong to the space group P422, with unit-cell parameters a = b = 100.7, c = 150.9 Angstrom. Using highly focused synchrotron radiation from the EMBL/ESRF beamline ID13 (Grenoble, France), diffraction to at least 3.2 Angstrom could be achieved. A selenomethionyl derivative of the protein was prepared and crystallized for future multiwavelength anomalous diffraction (MAD) experiments
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15kD protein.
No full textThe U5 small ribonucleoprotein particle (snRNP) contains various proteins involved in catalytic activities mediating conformational rearrangements of the spliceosome. We have isolated and characterized the evolutionarily highly conserved human U5 snRNP-specific protein U5-15kD. The crystal structure of U5-15kD determined at 1.4 Angstrom resolution revealed a thioredoxin-like fold and represents the first structure of a U5 snRNP-specific protein known so far. With respect to human thioredoxin the U5-15kD protein contains 37 additional residues causing structural changes which most likely form putative binding sites for other spliceosomal proteins or RNA. Moreover, a novel intramolecular disulfide bond replaces the canonical one found in the thioredoxin family. Even though U5-15kD appears to lack protein disulfide isomerase activity, it is strictly required for pre-mRNA splicing in vivo as we demonstrate by genetic depletion of its ortholog in Saccharomyces cerevisiae. Our data suggest that the previously reported involvement of its Schizosaccharomyces pombe ortholog Dim1p in cell cycle regulation is a consequence of its essential role in pre-mRNA splicing. (C) 1999 Academic Press
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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