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1.
  • Alzari, Pedro M., et al. (författare)
  • Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
  • 2006
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62, s. 1103-1113
  • Tidskriftsartikel (refereegranskat)abstract
    • The implementation of high- throughput ( HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe ( SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non- ligation- based cloning techniques used, namely Gateway, ligation- indendent cloning of PCR products ( LIC- PCR) and In- Fusion, with LIC- PCR emerging as the most cost- effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library- based screening for soluble constructs and parallel small- scale high- density fermentation.
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2.
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3.
  • Aricescu, A R, et al. (författare)
  • Eukaryotic expression: developments for structural proteomics.
  • 2006
  • Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 62, s. 1114-1124
  • Tidskriftsartikel (refereegranskat)abstract
    • The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
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4.
  • Backmark, Anna, 1979, et al. (författare)
  • Affinity tags can reduce merohedral twinning of membrane protein crystals
  • 2008
  • Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
  • Tidskriftsartikel (refereegranskat)abstract
    • This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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5.
  • Björkelid, Christofer, et al. (författare)
  • Structural and functional studies of mycobacterial IspD enzymes
  • 2011
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 67, s. 403-414
  • Tidskriftsartikel (refereegranskat)abstract
    • A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.
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6.
  • Björkelid, Christofer, et al. (författare)
  • Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
  • 2012
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68, s. 134-143
  • Tidskriftsartikel (refereegranskat)abstract
    • A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.
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7.
  • Castell, Alina, et al. (författare)
  • Structural analysis of mycobacterial branched-chain aminotransferase : implications for inhibitor design
  • 2010
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 66, s. 549-557
  • Tidskriftsartikel (refereegranskat)abstract
    • The branched-chain aminotransferase (BCAT) of Mycobacterium tuberculosis has been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5'-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features of M. tuberculosis BCAT, X-ray structural and functional analyses of the closely related BCAT from M. smegmatis were carried out. The crystal structures include the apo form at 2.2 angstrom resolution and a 1.9 angstrom structure of the holo form cocrystallized with the inhibitor O-benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor.
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8.
  • Charavgi, Maria-Despoina, et al. (författare)
  • The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst
  • 2013
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69:1, s. 63-73
  • Tidskriftsartikel (refereegranskat)abstract
    • The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-[beta]-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an [alpha]/[beta]-hydrolase fold with a three-layer [alpha][beta][alpha]-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.
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9.
  • Cheeseman, J. D., et al. (författare)
  • Structure of an aryl esterase from Pseudomonas fluorescens
  • 2004
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 60:7, s. 1237-1243
  • Tidskriftsartikel (refereegranskat)abstract
    • The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 Å by X-ray diffraction and shows a characteristic α/β-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 Cα atoms between PFE and its five closest structural neighbors averaging 0.8 Å. PFE has far less similarity (r.m.s. deviation in 218 Cα atoms of 5.0 Å) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
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10.
  • Chen, Yang, et al. (författare)
  • Structure of AadA from Salmonella enterica : a monomeric aminoglycoside (3'')(9) adenyltransferase
  • 2015
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71, s. 2267-2277
  • Tidskriftsartikel (refereegranskat)abstract
    • Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.
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11.
  • Cohen, Serge X., et al. (författare)
  • Towards complete validated models in the next generation of ARP/wARP
  • 2004
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 60:Pt 12 Pt 1, s. 2222-9
  • Tidskriftsartikel (refereegranskat)abstract
    • The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.
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12.
  • Dimarogona, M, et al. (författare)
  • The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft
  • 2012
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:7, s. 735-742
  • Tidskriftsartikel (refereegranskat)abstract
    • Xylanase enzymes have been the focus of considerable research in recent decades owing to their extensive use in a variety of biotechnological applications. Previous structural studies of a number of GH10 xylanases revealed that all GH10 family members have the (β/α)8-barrel fold and their catalytic site is conserved. The structure of a new GH10 xylanase from Fusarium oxysporum (FoXyn10a) was determined at 1.94 Å resolution from crystals belonging to the tetragonal space group P41212 with five molecules per asymmetric unit. Comparison of the structure of FoXyn10a with previously determined structures of GH10 family members indicated that most of the differences were located in the loop regions between the ordered secondary-structure elements of the barrel, as expected. However, alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand β6b and helix 6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme.
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13.
  • Dobritzsch, Doreen, 1972-, et al. (författare)
  • Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri
  • 2003
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 59:Pt 7, s. 1267-1269
  • Tidskriftsartikel (refereegranskat)abstract
    • In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.
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14.
  • Dobritzsch, Doreen, 1972-, et al. (författare)
  • Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase
  • 2001
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 57:Pt 1, s. 153-155
  • Tidskriftsartikel (refereegranskat)abstract
    • Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.
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15.
  • Ekström, Fredrik, 1973-, et al. (författare)
  • Crystallization of the actin-binding domain of human alpha-actinin : analysis of microcrystals of SeMet-labelled protein
  • 2003
  • Ingår i: Acta Crystallographica Section D. - : Blackwell Munksgaard. - 0907-4449 .- 1399-0047. ; 59:Pt 4, s. 724-726
  • Tidskriftsartikel (refereegranskat)abstract
    • Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.
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16.
  • Espaillat, Akbar, et al. (författare)
  • Structural basis for the broad specificity of a new family of amino-acid racemases
  • 2014
  • Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 70, s. 79-90
  • Tidskriftsartikel (refereegranskat)abstract
    • Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.
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17.
  • Fogg, M. J., et al. (författare)
  • Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens
  • 2006
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62:10, s. 1196-1207
  • Tidskriftsartikel (refereegranskat)abstract
    • The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
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18.
  • Frankaer, C. G., et al. (författare)
  • The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy
  • 2012
  • Ingår i: Acta Crystallographica Section D-Biological Crystallography. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 68, s. 1259-1271
  • Tidskriftsartikel (refereegranskat)abstract
    • The crystal structures of three conformations, T6, T3R3 and R6, of bovine insulin were solved at 1.40, 1.30 and 1.80 angstrom resolution, respectively. All conformations crystallized in space group R3. In contrast to the T6 and T3R3 structures, different conformations of the N-terminal B-chain residue PheB1 were observed in the R6 insulin structure, resulting in an eightfold doubling of the unit-cell volume upon cooling. The zinc coordination in each conformation was studied by X-ray absorption spectroscopy (XAS), including both EXAFS and XANES. Zinc adopts a tetrahedral coordination in all R3 sites and an octahedral coordination in T3 sites. The coordination distances were refined from XAS with a standard deviation of <0.01 angstrom. In contrast to the distances determined from the medium-resolution crystal structures, the XAS results were in good agreement with similar coordination geometries found in small molecules, as well as in other high-resolution insulin structures. As the radiation dose for XRD experiments is two orders of magnitude higher compared with that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage that affected the zinc coordination in the T3 sites in particular. Furthermore, XANES spectra for the zinc sites in T6 and R6 insulin were successfully calculated using finite difference methods and the bond distances and angles were optimized from a quantitative XANES analysis.
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19.
  • Friemann, Rosmarie, et al. (författare)
  • Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system
  • 2009
  • Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 65, s. 24-33
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
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20.
  • Grahn, Elin, et al. (författare)
  • Crystallization and preliminary X-ray crystallographic studies of a lectin from the mushroom Marasmius oreades
  • 2004
  • Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 60:11, s. 2038-2039
  • Tidskriftsartikel (refereegranskat)abstract
    • The Marasmius oreades agglutinin (MOA) recognizes blood group B oligosaccharides. This mushroom lectin belongs to the ricin superfamily and is currently the only lectin known with exclusive specificity for Galα1,3Gal-structures, as occur in the subterminally fucosylated blood group B epitope Galα1,3(Fucα1,2)Galβ1,4GlcNAc (MOA's preferred ligand) or without fucosylation in the xenotransplantation epitope. MOA has been co-crystallized with the linear blood group B trisaccharide Galα1,3Galβ1,4GlcNAc using the hanging-drop vapour-diffusion technique at room temperature. MOA crystals were grown in the presence of ammonium formate and HEPES buffer. A 3.0 Å data set has been collected. Preliminary analysis of the X-ray data is consistent with space group P3 1 or P3 2 and unit-cell parameters a = b = 105, c = 113 Å, with two dimers per asymmetric unit. © 2004 International Union of Crystallography.
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21.
  • Haddad Momeni, Majid, et al. (författare)
  • Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea
  • 2014
  • Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 70, s. 2356-2366
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10 degrees C higher melting temperature (T-m of 72.5 degrees C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65 degrees C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 angstrom resolution; R-work and R-free of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.
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22.
  • Hasse, Dirk, et al. (författare)
  • Structure of Arabidopsis thaliana Rubisco activase
  • 2015
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71:Pt 4, s. 800-808
  • Tidskriftsartikel (refereegranskat)abstract
    • The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase from Arabidopsis thaliana is presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.
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23.
  • Hällberg, B. Martin, et al. (författare)
  • Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor
  • 2004
  • Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 60, s. 197-199
  • Tidskriftsartikel (refereegranskat)abstract
    • Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H2O2 for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl2 and Ta6Br12. They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 Angstrom, beta = 90.85degrees. X-ray diffraction data to 2.0 Angstrom resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.
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24.
  • Ingvarsson, Henrik, et al. (författare)
  • Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1
  • 2007
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63:2, s. 249-259
  • Tidskriftsartikel (refereegranskat)abstract
    • Mycobacterium tuberculosis caseinolytic protease ClpP1 (Mt ClpP1) is a self-compartmentalized protease consisting of two heptameric rings stacked on top of each other, thus enclosing a catalytic chamber. Within the chamber, which can be reached through two axial pores, each of the 14 identical monomers possesses a serine protease active site. The unfolding and translocation of substrates into the chamber are mediated by associated hexameric ATPases covering the axial pores. Three crystal structures of Mt ClpP1, determined by molecular replacement, are presented in this study. Two of the models were refined to a resolution of 2.6 A and the third to 3.0 A. It was found that disorder in the handle domain affects the formation and configuration of the tetradecamer and results in condensed structures with larger equatorial pores when compared with ClpPs from other species. Additionally, this disorder accompanies conformational changes of the residues in the catalytic triad. The models also reveal structural differences within the N-terminal hairpin-loop domain, which possibly reflect the significant differences in amino-acid sequence between Mt ClpP1 and other ClpP homologues in this region.
  •  
25.
  • Jacobson, Frida, 1975, et al. (författare)
  • High resolution X-ray structures of the oxidised and reduced forms of nitrite reductase from Rhodobacter sphaeroides 2.4.3
  • 2005
  • Ingår i: Acta Crystallography D. - 0907-4449 .- 1399-0047. ; 61, s. 1190-1198
  • Tidskriftsartikel (refereegranskat)abstract
    • Nitrite reductase is an enzyme operating in the denitrification pathway which catalyses the conversion of nitrite (NO2(-)) to gaseous nitric oxide (NO). Here, crystal structures of the oxidized and reduced forms of the copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3 are presented at 1.74 and 1.85 A resolution, respectively. Whereas the structure of the enzyme is very similar to those of other copper-containing nitrite reductases, folding as a trimer and containing two copper sites per monomer, the structures reported here enable conformational differences between the oxidized and reduced forms of the enzyme to be identified. In the type 1 copper site, a rotational perturbation of the side chain of the copper ligand Met182 occurs upon reduction. At the type 2 copper site, a dual conformation of the catalytic residue His287 is observed in the oxidized structure but is lacking in the reduced structure, such that the interactions of the oxidized type 2 copper ion can be regarded as adopting octahedral geometry. These findings shed light on the structural mechanism of the reduction of a copper-bound nitrite to nitric oxide and water.
  •  
26.
  • Jansson, Anna M., et al. (författare)
  • Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector
  • 2009
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 65, s. 796-803
  • Tidskriftsartikel (refereegranskat)abstract
    • The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-l-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.
  •  
27.
  • Johansson, P., et al. (författare)
  • Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides
  • 2003
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 59, s. 535-537
  • Tidskriftsartikel (refereegranskat)abstract
    • Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls. Recombinant XET from poplar has been purified from a Pichia pastoris expression system and crystallized. Two different crystal forms were obtained by vapour diffusion from potassium sodium tartrate and from an imidazole buffer using sodium acetate as a precipitant. Data were collected from these crystal forms to 3.5 and 2.1 Angstrom resolution, respectively. The first crystal form was found to belong to space group P3(1)21 or P3(2)21 (unit-cell parameters a = 98.6, b = 98.6, c = 98.5 Angstrom) and the second crystal form to space group P6(3) (unit-cell parameters a = 188.7, b = 188.7, c = 46.1 Angstrom).
  •  
28.
  • Kleywegt, Gerard J. (författare)
  • Crystallographic refinement of ligand complexes
  • 2007
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63:1, s. 94-100
  • Tidskriftsartikel (refereegranskat)abstract
    • Model building and refinement of complexes between biomacromolecules and small molecules requires sensible starting coordinates as well as the specification of restraint sets for all but the most common non-macromolecular entities. Here, it is described why this is necessary, how it can be accomplished and what pitfalls need to be avoided in order to produce chemically plausible models of the low-molecular-weight entities. A number of programs, servers, databases and other resources that can be of assistance in the process are also discussed.
  •  
29.
  • Kleywegt, Gerard J. (författare)
  • On vital aid : the why, what and how of validation
  • 2009
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 65:Pt 2, s. 134-139
  • Tidskriftsartikel (refereegranskat)abstract
    • Limitations to the data and subjectivity in the structure-determination process may cause errors in macromolecular crystal structures. Appropriate validation techniques may be used to reveal problems in structures, ideally before they are analysed, published or deposited. Additionally, such techniques may be used a posteriori to assess the (relative) merits of a model by potential users. Weak validation methods and statistics assess how well a model reproduces the information that was used in its construction (i.e. experimental data and prior knowledge). Strong methods and statistics, on the other hand, test how well a model predicts data or information that were not used in the structure-determination process. These may be data that were excluded from the process on purpose, general knowledge about macromolecular structure, information about the biological role and biochemical activity of the molecule under study or its mutants or complexes and predictions that are based on the model and that can be tested experimentally.
  •  
30.
  • Kleywegt, Gerard J (författare)
  • Separating model optimization and model validation in statistical cross-validation as applied to crystallography.
  • 2007
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63:9, s. 939-940
  • Tidskriftsartikel (refereegranskat)abstract
    • Statistical cross-validation has become an integral part of the model-refinement process in macromolecular crystallography. However, the test set of reflections, for which the free R value is calculated, is used both to optimize the parameterization of the structure model and to validate the model itself. This practice could introduce bias and diminish the value of R(free) as an independent check of model quality. It is proposed here that by introducing a dormant hold-out set of reflections, any problems with such bias can be avoided. This procedure requires only a small modification of the standard cross-validation protocol.
  •  
31.
  • Kleywegt, Gerard J, et al. (författare)
  • The Uppsala Electron-Density Server
  • 2004
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 60:Pt 12 Pt 1, s. 2240-2249
  • Tidskriftsartikel (refereegranskat)abstract
    • The Uppsala Electron Density Server (EDS; http://eds.bmc.uu.se/) is a web-based facility that provides access to electron-density maps and statistics concerning the fit of crystal structures and their maps. Maps are available for approximately 87% of the crystallographic Protein Data Bank (PDB) entries for which structure factors have been deposited and for which straightforward map calculations succeed in reproducing the published R value to within five percentage points. Here, an account is provided of the methods that are used to generate the information contained in the server. Some of the problems that are encountered in the map-generation process as well as some spin-offs of the project are also discussed.
  •  
32.
  • Kleywegt, Gerard J., et al. (författare)
  • ValLigURL : a server for ligand-structure comparison and validation
  • 2007
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63, s. 935-938
  • Tidskriftsartikel (refereegranskat)abstract
    • A new web-based tool called ValLigURL is described. It can be used by practising crystallographers to validate the geometry of a ligand and to compare the conformation of a ligand with all instances of that ligand in the structural database (wwPDB). In addition, it can be used by structural bioinformaticians to survey the quality or conformational diversity of any ligand across the entire structural database. The server is freely accessible at the URL http://eds.bmc.uu.se/eds/valligurl.php.
  •  
33.
  • Knight, Stefan D. (författare)
  • RSPS version 4.0: : a semi-interactive vector-search program for solving heavy-atom derivatives
  • 2000
  • Ingår i: Acta Crystallographica Section D. - : MUNKSGAARD INT PUBL LTD. - 0907-4449 .- 1399-0047. ; 56:1, s. 42-47
  • Tidskriftsartikel (refereegranskat)abstract
    • A program for inspection and interpretation of the Patterson function is described. The program is mainly intended for finding heavy-atom positions from difference Patterson maps, but may also be used to locate molecules with non-crystallographic symmetry when the local axis is nearly parallel to a crystallographic symmetry axis. Options are available for vector-based methods to locate heavy-atom sites, for finding sets from a list of possible heavy-atom positions and for checking of potential solutions. Both crystallographic and non-crystallographic symmetry may be used, either independently or in conjunction.
  •  
34.
  • Kumar, Atul, et al. (författare)
  • The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis.
  • 2013
  • Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 69:Pt 12, s. 2543-54
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial N-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond between N-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 of Mycobacterium tuberculosis has been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed in Bartonella henselae amidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, an N-acetylmuramoyl-L-alanine amidase from M. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.
  •  
35.
  • Lindås, Ann-Christin, et al. (författare)
  • Structure of crenactin, an archaeal actin homologue active at 90 degrees C
  • 2014
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 70, s. 492-500
  • Tidskriftsartikel (refereegranskat)abstract
    • The crystal structure of the archaeal actin, crenactin, from the rod-shaped hyperthermophilic (optimal growth at 90 degrees C) crenarchaeon Pyrobaculum calidifontis is reported at 3.35 angstrom resolution. Despite low amino-acid sequence identity, the three-dimensional structure of the protein monomer is highly similar to those of eukaryotic actin and the bacterial MreB protein. Crenactin-specific features are also evident, as well as elements that are shared between crenactin and eukaryotic actin but are not found in MreB. In the crystal, crenactin monomers form right-handed helices, demonstrating that the protein is capable of forming filament-like structures. Monomer interactions in the helix, as well as interactions between crenactin and ADP in the nucleotide-binding pocket, are resolved at the atomic level and compared with those of actin and MreB. The results provide insights into the structural and functional properties of a heat-stable archaeal actin and contribute to the understanding of the evolution of actin-family proteins in the three domains of life.
  •  
36.
  • Lohkamp, Bernhard, et al. (författare)
  • A mixture of fortunes : the curious determination of the structure of Escherichia coli BL21 Gab protein
  • 2008
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 64:Pt 4, s. 407-415
  • Tidskriftsartikel (refereegranskat)abstract
    • In protein crystallography, monodisperse protein samples of high purity are usually required in order to obtain diffraction-quality crystals. Here, crystals were reproducibly grown from a protein sample before its homogeneity had been determined. The sample was obtained after the first attempt to purify a recombinant target protein from an Escherichia coli cell lysate. Subsequent analysis revealed that it was a mixture of about 50 different proteins with no predominant species. Diffraction data were collected to 2.1 A and the space group was identified as I422. A molecular-replacement search with models of the expected target did not give a solution, which suggested that a contaminating E. coli protein had been crystallized. A PDB search revealed 256 structures determined in space group I422, of which 14 are E. coli proteins and two have unit-cell parameters similar to those observed. Molecular replacement with these structures showed a clear solution for one of them, the Gab protein. The structure is presented and compared with the deposited structure, from which it shows small but significant differences. The refined model contains bicine and sulfate as bound ligands, which provide insights into possible substrate-binding sites.
  •  
37.
  • Meining, Winfried, et al. (författare)
  • Crystallization and preliminary crystallographic analysis of the recombinant N-terminal domain of riboflavin synthase
  • 2001
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 57, s. 1296-1299
  • Tidskriftsartikel (refereegranskat)abstract
    • Riboflavin synthase catalyzes the final step in the biosynthesis of riboflavin. Animals and humans lack this enzyme, whereas many bacteria and certain yeasts are absolutely dependent on endogenous riboflavin synthesis. Riboflavin synthase is therefore an attractive target for chemotherapy. The N-terminal domain of riboflavin synthase forms a dimer in solution and is capable of strongly binding riboflavin. It can serve as a model for the binding site of the native enzyme. Structural information obtained from this domain at high resolution will be helpful in the determination of the binding mode of riboflavin and thus for the development of antimicrobial drugs. Here, the crystallization and preliminary crystallographic analysis of the N-terminal domain of riboflavin synthase are reported. The crystals belong to the space group C222(1), with unit-cell parameters a = 50.3, b = 104.7, c = 85.3 Angstrom, alpha = beta = gamma = 90 degrees, and diffract to 2.6 Angstrom resolution.
  •  
38.
  • Novotny, Marian, et al. (författare)
  • On the precision of calculated solvent-accessible surface areas
  • 2007
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63:2, s. 270-274
  • Tidskriftsartikel (refereegranskat)abstract
    • The fact that protein structures are dynamic by nature and that structure models determined by X-ray crystallography, electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy have limited accuracy limits the precision with which derived properties can be reported. Here, the issue of the precision of calculated solvent-accessible surface areas (ASAs) is addressed. A number of protein structures of different sizes were selected and the effect of random shifts applied to the atomic coordinates on ASA values was investigated. Standard deviations of the ASA calculations were found to range from ∼10 to ∼80  Å2. Similar values are obtained for a handful of cases in the Protein Data Bank (PDB) where `ensembles' of crystal structures were refined against the same data set. The ASA values for 69 hen egg-white lysozyme structures were calculated and a standard deviation of the ASA of 81  Å2 was obtained (the average ASA value was 6571  Å2). The calculated ASA values do not show any correlation with factors such as resolution or overall temperature factors. A molecular-dynamics (MD) trajectory of lysozyme was also analysed. The ASA values during the simulation covered a range of more than 800  Å2. If different programs are used, the ASA values obtained for one small protein show a spread of almost 600  Å2. These results suggest that in most cases reporting ASA values with a precision better than 10  Å2 is probably not realistic and a precision of 50–100  Å2 would seem prudent. The precision of buried surface-area calculations for complexes is also discussed.
  •  
39.
  • Osipov, Evgeny, et al. (författare)
  • Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties
  • 2014
  • Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography. - 0907-4449 .- 1399-0047. ; 70:11, s. 2913-2923
  • Tidskriftsartikel (refereegranskat)abstract
    • Laccases are members of a large family of multicopper oxidases that catalyze the oxidn. of a wide range of org. and inorg. substrates accompanied by the redn. of dioxygen to water. These enzymes contain four Cu atoms per mol. organized into three sites: T1, T2 and T3. In all laccases, the T1 copper ion is coordinated by two histidines and one cysteine in the equatorial plane and is covered by the side chains of hydrophobic residues in the axial positions. The redox potential of the T1 copper ion influences the enzymic reaction and is detd. by the nature of the axial ligands and the structure of the second coordination sphere. In this work, the laccase from the ascomycete Botrytis aclada was studied, which contains conserved Ile491 and nonconserved Leu499 residues in the axial positions. The three-​dimensional structures of the wild-​type enzyme and the L499M mutant were detd. by X-​ray crystallog. at 1.7 Å resoln. Crystals suitable for X-​ray anal. could only be grown after deglycosylation. Both structures did not contain the T2 copper ion. The catalytic properties of the enzyme were characterized and the redox potentials of both enzyme forms were detd.: E 0 = 720 and 580 mV for the wild-​type enzyme and the mutant, resp. Since the structures of the wild-​type and mutant forms are very similar, the change in the redox potential can be related to the L499M mutation in the T1 site of the enzyme.
  •  
40.
  • Oswald, Christine, et al. (författare)
  • Crystallization and preliminary crystallographic analysis of the NAD(H)-binding domain of Escherichia coli transhydrogenase
  • 2004
  • Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 60:4, s. 743-745
  • Tidskriftsartikel (refereegranskat)abstract
    • Transhydrogenase is a proton-pumping membrane protein that is required for the cellular regeneration of NADPH. The NAD(H)-binding domain (domain I) of transhydrogenase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique at room temperature. The crystals, which were grown from PEG 4000 and ammonium acetate in citrate buffer, belong to the triclinic space group P1, with unit-cell parameters a = 38.8, b = 66.8, c = 76.4 Å, α = 67.5, β = 80.8, γ = 81.5°. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The crystals contain one dimer of transhydrogenase domain I per asymmetric unit. © 2004 International Union of Crystallography. Printed in Denmark - all rights reserved.
  •  
41.
  • Persson, Karina, 1969- (författare)
  • Structure of the sortase AcSrtC-1 from Actinomyces oris
  • 2011
  • Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography. - 0907-4449 .- 1399-0047. ; 67, s. 212-217
  • Tidskriftsartikel (refereegranskat)abstract
    • The crystal structure of the sortase AcSrtC-1 from the oral microorganism Actinomyces oris has been determined to 2.4 Å resolution. AcSrtC-1 is a cysteine transpeptidase that is responsible for the formation of fimbriae by the polymerization of a shaft protein. Similar to other pili-associated sortases, the AcSrtC-1 active site is protected by a flexible lid. The asymmetric unit contains five AcSrtC-1 molecules and their catalytic Cys-His-Arg triads are trapped in two different conformations. It is also shown that the thermostability of the enzyme is increased by the presence of calcium.
  •  
42.
  • Persson, Magnus, et al. (författare)
  • Prr1 Coat Protein Binding to its Translational Operator
  • 2013
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 367-372
  • Tidskriftsartikel (refereegranskat)abstract
    • In small RNA bacteriophages, the genomic RNA binds to the coat proteins when the viral capsid assembles. This is achieved through sequence-specific interactions between a coat-protein dimer and an RNA stem-loop that includes the start codon for the replicase gene. The structure of virus-like particles of the small RNA phage PRR1 bound to an RNAsegment corresponding to this stem-loop has been solved and the binding was compared with the related, and better investigated, phage MS2. The overall conformation of the RNA is found to be similar and the residues that are involved in RNA binding in PRR1 are the same as in MS2. The arrangement of the nucleotide bases in the loop of the stem-loop is different, leading to a difference in the stacking at the conserved Tyr86, which is equivalent to Tyr85 in MS2.
  •  
43.
  • Read, Randy J, et al. (författare)
  • Case-controlled structure validation
  • 2009
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 65:Pt 2, s. 140-147
  • Tidskriftsartikel (refereegranskat)abstract
    • Although many factors influence the quality of a macromolecular crystal structure, validation criteria are usually only calibrated using one of these factors, the resolution. For many purposes this is sufficient, but there are times when one wishes to compare one set of structures with another and the comparison may be invalidated by systematic differences between the sets in factors other than resolution. This problem can be circumvented by borrowing from medicine the idea of the case-matched control: each structure of interest is matched with a control structure that has similar values for all relevant factors considered in this study. In addition to resolution, these include the size of the structure (as measured by the volume of the asymmetric unit) and the year of deposition. This approach has been applied to address two questions: whether structures from structural genomics efforts reach the same level of quality as structures from traditional sources and whether the impact factor of the journal in which a structure is published correlates with structure quality. In both cases, once factors influencing quality have been controlled in the comparison, there is little evidence for a systematic difference in quality.
  •  
44.
  • Scian, Michele, et al. (författare)
  • Comparison of epsilon- and delta-class glutathione S-transferases : the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster
  • 2015
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71, s. 2089-2098
  • Tidskriftsartikel (refereegranskat)abstract
    • Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta-and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5 angstrom resolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon-and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.
  •  
45.
  • Selmer, Maria, et al. (författare)
  • Ribosome engineering to promote new crystal forms
  • 2012
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:5, s. 578-583
  • Tidskriftsartikel (refereegranskat)abstract
    • Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in similar to the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.
  •  
46.
  • Sierra, Raymond G., et al. (författare)
  • Nanoflow electrospinning serial femtosecond crystallography
  • 2012
  • Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 68, s. 1584-1587
  • Tidskriftsartikel (refereegranskat)abstract
    • An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 mu l min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 mu l min(-1) and diffracted to beyond 4 angstrom resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 mu g of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
  •  
47.
  • Stepper, Judith, et al. (författare)
  • Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-beta-glucosidase SPy1599
  • 2013
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 16-23
  • Tidskriftsartikel (refereegranskat)abstract
    • The group A streptococcus Streptococcus pyogenes is the causative agent of a wide spectrum of invasive infections, including necrotizing fasciitis, scarlet fever and toxic shock syndrome. In the context of its carbohydrate chemistry, it is interesting that S. pyogenes (in this work strain M1 GAS SF370) displays a spectrum of oligosaccharide-processing enzymes that are located in close proximity on the genome but that the in vivo function of these proteins remains unknown. These proteins include different sugar transporters (SPy1593 and SPy1595), both GH125 alpha-1,6- and GH38 alpha-1,3-mannosidases (SPy1603 and SPy1604), a GH84 beta-hexosaminidase (SPy1600) and a putative GH2 beta-galactosidase (SPy1586), as well as SPy1599, a family GH1 'putative beta-glucosidase'. Here, the solution of the three-dimensional structure of SPy1599 in a number of crystal forms complicated by unusual crystallographic twinning is reported. The structure is a classical (beta/alpha)(g)-barrel, consistent with CAZy family GH1 and other members of the GH-A clan. SPy1599 has been annotated in sequence depositions as a beta-glucosidase (EC 3.2.1.21), but no such activity could be found; instead, three-dimensional structural overlaps with other enzymes of known function suggested that SPy1599 contains a phosphate-binding pocket in the active site and has possible 6-phospho-beta-glycosidase activity. Subsequent kinetic analysis indeed showed that SPy1599 has 6-phospho-beta-glucosidase (EC 3.2.1.86) activity. These data suggest that SPy1599 is involved in the intracellular degradation of 6-phosphoglycosides, which are likely to originate from import through one of the organism's many phosphoenolpyruvate phosphotransfer systems (PEP-PTSs).
  •  
48.
  • Urzhumtsev, Alexandre, et al. (författare)
  • Ultralow-resolution ab initio phasing of filamentous proteins : crystals from a six-Ig fragment of titin as a case study.
  • 2008
  • Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell Publishing Inc.. - 0907-4449 .- 1399-0047. ; 64:Pt 5, s. 478-486
  • Tidskriftsartikel (refereegranskat)abstract
    • Low-resolution diffraction data (resolution below 12 angstroms) from crystals of a filamentous six-Ig fragment of titin, I65-I70, were used in ab initio phasing with the aim of calculating its lattice packing and molecular envelope. Filamentous molecules, characterized by marked anisometry and idiosyncratic crystal lattices, have not been addressed before using this methodology. In this study, low-resolution phasing (19-122 angstroms) successfully identified the region of the unit cell occupied by the molecule. Phase extension to a higher resolution (12 angstroms) yielded regions of high density that corresponded either to the positions of individual Ig domains or to zones of dense intermolecular contacts, hindering the identification of individual domains and the interpretation of electron-density maps in terms of a molecular model. This problem resulted from the acutely uneven packing of the molecules in the crystal and it was further accentuated by the presence of partially disordered regions in the molecule. Addition of low-resolution reflections with phases computed ab initio to those obtained experimentally using MIRAS improved the initial electron-density maps of the atomic model, demonstrating the generic utility of low-resolution phases for the structure-elucidation process, even when individual molecules cannot be resolved in the lattice.
  •  
49.
  • Valegård, Karin, et al. (författare)
  • Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway
  • 2013
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 1567-1579
  • Tidskriftsartikel (refereegranskat)abstract
    • Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/beta-lactamase-type fold with highest structural similarity to the class A beta-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show beta-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, beta-lactamases and d-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.
  •  
50.
  • Vasur, Jonas, et al. (författare)
  • X-ray crystallographic native sulfur SAD structure determination of laminarinase Lam16A from Phanerochaete chrysosporium
  • 2006
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62:11, s. 1422-1429
  • Tidskriftsartikel (refereegranskat)abstract
    • Laminarinase Lam16A from Phanerochaete chrysosporium was recombinantly expressed in Pichia pastoris, crystallized and the structure was solved at 1.34 A resolution using native sulfur SAD X-ray crystallography. It is the first structure of a non-specific 1,3(4)-beta-D-glucanase from glycoside hydrolase family 16 (GH16). P. chrysosporium is a wood-degrading basidiomycete fungus and Lam16A is the predominant extracellular protein expressed when laminarin is used as the sole carbon source. The protein folds into a curved beta-sandwich homologous to those of other known GH16 enzyme structures (especially kappa-carrageenase from Pseudo-alteromonas carrageenovora and beta-agarase from Zobelia galactanivorans). A notable likeness is also evident with the related glycoside hydrolase family 7 (GH7) enzymes. A mammalian lectin, p58/ERGIC, as well as polysaccharide lyase (PL7) enzymes also showed significant similarity to Lam16A. The enzyme has two potential N-glycosylation sites. One such site, at Asn43, displayed a branched heptasaccharide sufficiently stabilized to be interpreted from the X-ray diffraction data. The other N-glycosylation motif was found close to the catalytic centre and is evidently not glycosylated.
  •  
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