| 1. |
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| 2. |
- Björkelid, Christofer, et al.
(author)
-
Structural and functional studies of mycobacterial IspD enzymes
- 2011
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 67, s. 403-414
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Journal article (peer-reviewed)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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| 3. |
- Björkelid, Christofer, et al.
(author)
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Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
- 2012
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68, s. 134-143
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Journal article (peer-reviewed)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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| 4. |
- Castell, Alina, et al.
(author)
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Structural analysis of mycobacterial branched-chain aminotransferase : implications for inhibitor design
- 2010
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 66, s. 549-557
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Journal article (peer-reviewed)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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| 5. |
- Charavgi, Maria-Despoina, et al.
(author)
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The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst
- 2013
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69:1, s. 63-73
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Journal article (peer-reviewed)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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| 6. |
- Dimarogona, M, et al.
(author)
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The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft
- 2012
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:7, s. 735-742
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Journal article (peer-reviewed)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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| 7. |
- Espaillat, Akbar, et al.
(author)
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Structural basis for the broad specificity of a new family of amino-acid racemases
- 2014
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In: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 70, s. 79-90
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Journal article (peer-reviewed)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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| 8. |
- Frankaer, C. G., et al.
(author)
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The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy
- 2012
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In: Acta Crystallographica Section D-Biological Crystallography. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 68, s. 1259-1271
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Journal article (peer-reviewed)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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| 9. |
- Haddad Momeni, Majid, et al.
(author)
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Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea
- 2014
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In: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 70, s. 2356-2366
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Journal article (peer-reviewed)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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| 10. |
- Kumar, Atul, et al.
(author)
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The structure of Rv3717 reveals a novel amidase from Mycobacterium tuberculosis.
- 2013
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In: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 69:Pt 12, s. 2543-54
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Journal article (peer-reviewed)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.
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| 11. |
- Lindås, Ann-Christin, et al.
(author)
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Structure of crenactin, an archaeal actin homologue active at 90 degrees C
- 2014
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 70, s. 492-500
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Journal article (peer-reviewed)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.
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| 12. |
- Osipov, Evgeny, et al.
(author)
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Effect of the L499M mutation of the ascomycetous Botrytis aclada laccase on redox potential and catalytic properties
- 2014
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In: Acta Crystallographica Section D. - : International Union of Crystallography. - 0907-4449 .- 1399-0047. ; 70:11, s. 2913-2923
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Journal article (peer-reviewed)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.
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| 13. |
- Persson, Karina, 1969-
(author)
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Structure of the sortase AcSrtC-1 from Actinomyces oris
- 2011
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In: Acta Crystallographica Section D. - : International Union of Crystallography. - 0907-4449 .- 1399-0047. ; 67, s. 212-217
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Journal article (peer-reviewed)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.
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| 14. |
- Persson, Magnus, et al.
(author)
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Prr1 Coat Protein Binding to its Translational Operator
- 2013
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 367-372
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Journal article (peer-reviewed)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.
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| 15. |
- Selmer, Maria, et al.
(author)
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Ribosome engineering to promote new crystal forms
- 2012
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:5, s. 578-583
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Journal article (peer-reviewed)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.
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| 16. |
- Sierra, Raymond G., et al.
(author)
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Nanoflow electrospinning serial femtosecond crystallography
- 2012
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In: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 68, s. 1584-1587
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Journal article (peer-reviewed)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.
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| 17. |
- Stepper, Judith, et al.
(author)
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Structure and activity of the Streptococcus pyogenes family GH1 6-phospho-beta-glucosidase SPy1599
- 2013
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 16-23
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Journal article (peer-reviewed)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).
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| 18. |
- Valegård, Karin, et al.
(author)
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Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway
- 2013
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In: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69, s. 1567-1579
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Journal article (peer-reviewed)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.
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| 19. |
- Awad, Wael, et al.
(author)
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Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration.
- 2013
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 69:Pt 12, s. 2524-2533
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Journal article (peer-reviewed)abstract
- The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.
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| 20. |
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| 21. |
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| 22. |
- Emsley, P, et al.
(author)
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Features and development of Coot
- 2010
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In: Acta crystallographica. Section D, Biological crystallography. - 1399-0047. ; 66:Pt 4, s. 486-501
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Journal article (peer-reviewed)
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| 23. |
- Frankaer, Christian Grundahl, et al.
(author)
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The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy
- 2012
-
In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 68, s. 1259-1271
-
Journal article (peer-reviewed)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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| 24. |
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| 25. |
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| 26. |
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