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1.	Bielecki, Johan, 1982, et al. (författare) Electrospray sample injection for single-particle imaging with x-ray lasers 2019 Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:5 Tidskriftsartikel (refereegranskat)abstract The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.
2.	Carlsson, Gunilla H., et al. (författare) The elusive ligand complexes of the DWARF14 strigolactone receptor 2018 Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 69:9, s. 2345-2354 Tidskriftsartikel (refereegranskat)abstract Strigolactones, a group of terpenoid lactones, control many aspects of plant growth and development, but the active forms of these plant hormones and their mode of action at the molecular level are still unknown. The strigolactone protein receptor is unusual because it has been shown to cleave the hormone and supposedly forms a covalent bond with the cleaved hormone fragment. This interaction is suggested to induce a conformational change in the receptor that primes it for subsequent interaction with partners in the signalling pathway. Substantial efforts have been invested into describing the interaction of synthetic strigolactone analogues with the receptor, resulting in a number of crystal structures. This investigation combines a re-evaluation of models in the Protein Data Bank with a search for new conditions that may permit the capture of a receptor-ligand complex. While weak difference density is frequently observed in the binding cavity, possibly due to a low-occupancy compound, the models often contain features not supported by the X-ray data. Thus, at this stage, we do not believe that any detailed deductions about the nature, conformation, or binding mode of the ligand can be made with any confidence.
3.	Daurer, Benedikt J., et al. (författare) Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses 2017 Ingår i: IUCrJ. - : INT UNION CRYSTALLOGRAPHY. - 2052-2525. ; 4, s. 251-262 Tidskriftsartikel (refereegranskat)abstract This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of similar to 40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from similar to 35 to similar to 300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 * 10(12) photons per mu m(2) per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
4.	Eisenhut, Marion, et al. (författare) The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria. 2006 Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 142:1 Tidskriftsartikel (refereegranskat)abstract The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.
5.	Ekeberg, Tomas, 1983-, et al. (författare) Observation of a single protein by ultrafast X-ray diffraction 2024 Ingår i: Light. - : Springer Nature. - 2095-5545 .- 2047-7538. ; 13:1 Tidskriftsartikel (refereegranskat)abstract The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
6.	Gorkhover, Tais, et al. (författare) Femtosecond X-ray Fourier holography imaging of free-flying nanoparticles 2018 Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 12:3, s. 150-153 Tidskriftsartikel (refereegranskat)abstract Ultrafast X-ray imaging on individual fragile specimens such as aerosols 1 , metastable particles 2 , superfluid quantum systems 3 and live biospecimens 4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined 4,5 . Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale.
7.	Hagemann, Martin, et al. (författare) Detection of a phage genome carrying a zonula occludens like toxin gene (zot) in clinical isolates of Stenotrophomonas maltophilia. 2006 Ingår i: Archives of Microbiology. - : Springer Science and Business Media LLC. - 0302-8933 .- 1432-072X. ; 185:6 Tidskriftsartikel (refereegranskat)abstract During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called phiSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage phiLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on phiSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.
8.	Hagemann, Martin, et al. (författare) The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol. 2008 Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 190:17 Tidskriftsartikel (refereegranskat)abstract The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.
9.	Hantke, Max F., et al. (författare) A data set from flash X-ray imaging of carboxysomes 2016 Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3 Tidskriftsartikel (refereegranskat)abstract Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
10.	Hantke, Max F., et al. (författare) High-throughput imaging of heterogeneous cell organelles with an X-ray laser 2014 Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949 Tidskriftsartikel (refereegranskat)abstract We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
11.	Hasse, Dirk, et al. (författare) Alternative splicing produces an H-protein with better substrate properties for the P-protein of glycine decarboxylase. 2009 Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:23 Tidskriftsartikel (refereegranskat)abstract Several thousand plant genes are known to produce multiple transcripts, but the precise function of most of the alternatively encoded proteins is not known. Alternative splicing has been reported for the H-protein subunit of glycine decarboxylase in the genus Flaveria. H-protein has no catalytic activity itself but is a substrate of the three enzymatically active subunits, P-, T- and L-protein. In C(4) species of Flaveria, two H-proteins originate from single genes in an organ-dependent manner. Here, we report on differences between the two alternative H-protein variants with respect to their interaction with the glycine-decarboxylating subunit, P-protein. Steady-state kinetic analyses of the alternative Flaveria H-proteins and artificially produced 'alternative' Arabidopsis H-proteins, using either pea mitochondrial matrix extracts or recombinant cyanobacterial P-protein, consistently demonstrate that the alternative insertion of two alanine residues at the N-terminus of the H-protein elevates the activity of P-protein by 20%in vitro, and could promote glycine decarboxylase activity in vivo.
12.	Hasse, Dirk, et al. (författare) Crystallization and preliminary X-ray diffraction analyses of the homodimeric glycine decarboxylase (P-protein) from the cyanobacterium Synechocystis sp. PCC 6803. 2010 Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 66:Pt 2, s. 187-191 Tidskriftsartikel (refereegranskat)abstract Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C(1) metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215 kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1 A were obtained using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.30, b = 135.81, c = 179.08 A.
13.	Hasse, Dirk, et al. (författare) Properties of recombinant glycine decarboxylase P- and H-protein subunits from the cyanobacterium Synechocystis sp. strain PCC 6803. 2007 Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 581:7 Tidskriftsartikel (refereegranskat)abstract The multi-enzyme complex glycine decarboxylase is important for one-carbon metabolism, essential for the photorespiratory glycolate cycle of plants, and comprises four different polypeptides, P-, H-, T-, and L-protein. We report on the production and properties of recombinant P-protein from the cyanobacterium Synechocystis and also describe features of recombinant H-protein from the same organism. The P-protein shows enzymatic activity with lipoylated H-protein and very low activity with H-apoprotein or lipoate as artificial cofactors. Its affinity towards glycine is unaffected by the presence and nature of the methyleneamine acceptor molecule. The cyanobacterial H-protein apparently forms stable dimers.
14.	Hasse, Dirk, et al. (författare) Structure and mechanism of piperideine-6-carboxylate dehydrogenase from Streptomyces clavuligerus 2019 Ingår i: Acta Crystallographica Section D. - : INT UNION CRYSTALLOGRAPHY. - 2059-7983. ; 75, s. 1107-1118 Tidskriftsartikel (refereegranskat)abstract The core of beta-lactam antibiotics originates from amino acids of primary metabolism in certain microorganisms. beta-Lactam-producing bacteria, including Streptomyces clavuligerus, synthesize the precursor of the amino acid alpha-aminoadipic acid by the catabolism of lysine in two steps. The second reaction, the oxidation of piperideine-6-carboxylate (or its open-chain form alpha-aminoadipate semialdehyde) to alpha-aminoadipic acid, is catalysed by the NAD(+)-dependent enzyme piperideine-6-carboxylate dehydrogenase (P6CDH). This structural study, focused on ligand binding and catalysis, presents structures of P6CDH from S. clavuligerus in its apo form and in complexes with the cofactor NAD(+), the product alpha-aminoadipic acid and a substrate analogue, picolinic acid. P6CDH adopts the common aldehyde dehydrogenase fold, consisting of NAD-binding, catalytic and oligomerization domains. The product binds in the oxyanion hole, close to the catalytic residue Cys299. Clear density is observed for the entire cofactor, including the nicotinamide riboside, in the binary complex. NAD(+) binds in an extended conformation with its nicotinamide ring overlapping with the binding site of the carboxylate group of the product, implying that the conformation of the cofactor may change during catalysis. The binding site of the substrate analogue overlaps with that of the product, suggesting that the cyclic form of the substrate, piperideine-6-carboxylate, may be accepted as a substrate by the enzyme. The catalytic mechanism and the roles of individual residues are discussed in light of these results.
15.	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.
16.	Hasse, Dirk, et al. (författare) Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp PCC 6803 Suggests a Mechanism for Redox Regulation 2013 Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:49, s. 35333-35345 Tidskriftsartikel (refereegranskat)abstract Glycine decarboxylase, or P-protein, is a pyridoxal 5-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an (2) homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys(972) in the C terminus and Cys(353) located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys(972) and Cys(353) by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353-972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.
17.	Krauss, Tobias, et al. (författare) Preventive medicine of von Hippel-Lindau disease-associated pancreatic neuroendocrine tumors 2018 Ingår i: Endocrine-Related Cancer. - : BIOSCIENTIFICA LTD. - 1351-0088 .- 1479-6821. ; 25:9, s. 783-793 Tidskriftsartikel (refereegranskat)abstract Pancreatic neuroendocrine tumors (PanNETs) are rare in von Hippel-Lindau disease (VHL) but cause serious morbidity and mortality. Management guidelines for VHL-PanNETs continue to be based on limited evidence, and survival data to guide surgical management are lacking. We established the European-American-Asian-VHL-PanNET-Registry to assess data for risks for metastases, survival and long-term outcomes to provide best management recommendations. Of 2330 VHL patients, 273 had a total of 484 PanNETs. Median age at diagnosis of PanNET was 35 years (range 10-75). Fifty-five (20%) patients had metastatic PanNETs. Metastatic PanNETs were significantly larger (median size 5 vs 2 cm; P < 0.001) and tumor volume doubling time (TVDT) was faster (22 vs 126 months; P = 0.001). All metastatic tumors were >= 2.8 cm. Codons 161 and 167 were hotspots for VHL germline mutations with enhanced risk for metastatic PanNETs. Multivariate prediction modeling disclosed maximum tumor diameter and TVDT as significant predictors for metastatic disease (positive and negative predictive values of 51% and 100% for diameter cut-off >= 2.8 cm, 44% and 91% for TVDT cut-off of <= 24 months). In 117 of 273 patients, PanNETs > 1.5 cm in diameter were operated. Ten-year survival was significantly longer in operated vs non-operated patients, in particular for PanNETs < 2.8 cm vs >= 2.8 cm (94% vs 85% by 10 years; P = 0.020; 80% vs 50% at 10 years; P = 0.030). This study demonstrates that patients with PanNET approaching the cut-off diameter of 2.8 cm should be operated. Mutations in exon 3, especially of codons 161/167 are at enhanced risk for metastatic PanNETs. Survival is significantly longer in operated non-metastatic VHL-PanNETs.
18.	Larsson, Anna M., et al. (författare) Crystal structures of β-carboxysome shell protein CcmP : ligand binding correlates with the closed or open central pore 2017 Ingår i: Journal of Experimental Botany. - : OXFORD UNIV PRESS. - 0022-0957 .- 1460-2431. ; 68:14, s. 3857-3867 Tidskriftsartikel (refereegranskat)abstract Cyanobacterial CO2 fixation is promoted by encapsulating and co-localizing the CO2-fixing enzymes within a protein shell, the carboxysome. A key feature of the carboxysome is its ability to control selectively the flux of metabolites in and out of the shell. The beta-carboxysome shell protein CcmP has been shown to form a double layer of pseudohexamers with a relatively large central pore (similar to 13 angstrom diameter), which may allow passage of larger metabolites such as the substrate for CO2 fixation, ribulose 1,5-bisphosphate, through the shell. Here we describe two crystal structures, at 1.45 angstrom and 1.65 angstrom resolution, of CcmP from Synechococcus elongatus PCC7942 (SeCcmP). The central pore of CcmP is open or closed at its ends, depending on the conformation of two conserved residues, Glu69 and Arg70. The presence of glycerol resulted in a pore that is open at one end and closed at the opposite end. When glycerol was omitted, both ends of the barrel became closed. A binding pocket at the interior of the barrel featured residual density with distinct differences in size and shape depending on the conformation, open or closed, of the central pore of SeCcmP, suggestive of a metabolite-driven mechanism for the gating of the pore.
19.	Lundholm, Ida V., et al. (författare) Considerations for three-dimensional image reconstruction from experimental data in coherent diffractive imaging 2018 Ingår i: IUCrJ. - : International Union of Crystallography. - 2052-2525. ; 5, s. 531-541 Tidskriftsartikel (refereegranskat)abstract Diffraction before destruction using X-ray free-electron lasers (XFELs) has the potential to determine radiation-damage-free structures without the need for crystallization. This article presents the three-dimensional reconstruction of the Melbournevirus from single-particle X-ray diffraction patterns collected at the LINAC Coherent Light Source (LCLS) as well as reconstructions from simulated data exploring the consequences of different kinds of experimental sources of noise. The reconstruction from experimental data suffers from a strong artifact in the center of the particle. This could be reproduced with simulated data by adding experimental background to the diffraction patterns. In those simulations, the relative density of the artifact increases linearly with background strength. This suggests that the artifact originates from the Fourier transform of the relatively flat background, concentrating all power in a central feature of limited extent. We support these findings by significantly reducing the artifact through background removal before the phase-retrieval step. Large amounts of blurring in the diffraction patterns were also found to introduce diffuse artifacts, which could easily be mistaken as biologically relevant features. Other sources of noise such as sample heterogeneity and variation of pulse energy did not significantly degrade the quality of the reconstructions. Larger data volumes, made possible by the recent inauguration of high repetition-rate XFELs, allow for increased signal-to-background ratio and provide a way to minimize these artifacts. The anticipated development of three-dimensional Fourier-volume-assembly algorithms which are background aware is an alternative and complementary solution, which maximizes the use of data.
20.	Menkveld, Albert J., et al. (författare) Nonstandard Errors 2024 Ingår i: JOURNAL OF FINANCE. - : Wiley-Blackwell. - 0022-1082 .- 1540-6261. ; 79:3, s. 2339-2390 Tidskriftsartikel (refereegranskat)abstract In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty-nonstandard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for more reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants.
21.	Munke, Anna (författare) Structural studies of small viruses using an X-ray Free Electron Laser 2016 Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract X-ray crystallography has since its introduction been the most successful technique for solving protein structures. Viruses however, often possess structural components such as fibrils, tails and envelopes that make them difficult or impossible to crystallize. To explore virus structures and the structural changes they undergo during host entry and infection, instrumental developments are required. X-ray Free Electron Lasers posses some advantages compared to conventional synchrotron sources, which enable experiments that previously were impossible. The femtosecond pulses and peak brilliance that exceeds synchrotrons by 109 facilitate recording of diffraction from nano/microcrystals and single particles before radiation damage takes place. The challenges for XFELs to reach its true potential in structural biology are nevertheless still many. During the technical and computational developments, using well-characterized reference samples is advantageous. In this thesis, the Rice Dwarf virus and MS2 bacteriophage have been used for single particle imaging and crystallography experiments using XFELs. These viruses are two of the smallest biological samples so far studied as single particles using this technique and the crystallography data of MS2 presented might serve as basis for solving the first high-resolution genome structure.Nanodiamonds, having a similar elemental composition as biological samples, could potentially serve as reference samples in XFEL studies. However, the biomedical field also has an interest in nanodiamonds, for drug delivery and as implant coating for example. Toxicity and biocompatibility is therefore a legitimate concern. Here, results from toxicity experiments of nanodiamonds on bacterial and zebrafish model organisms are presented.
22.	Rath, Asawari D., et al. (författare) Explosion dynamics of sucrose nanospheres monitored by time of flight spectrometry and coherent diffractive imaging at the split-and-delay beam line of the FLASH soft X-ray laser 2014 Ingår i: Optics Express. - 1094-4087. ; 22:23, s. 28914-28925 Tidskriftsartikel (refereegranskat)abstract We use a Mach-Zehnder type autocorrelator to split and delay XUV pulses from the FLASH soft X-ray laser for triggering and subsequently probing the explosion of aerosolised sugar balls. FLASH was running at 182 eV photon energy with pulses of 70 fs duration. The delay between the pump-probe pulses was varied between zero and 5 ps, and the pulses were focused to reach peak intensities above 1016 W/cm2 with an off-axis parabola. The direct pulse triggered the explosion of single aerosolised sucrose nano-particles, while the delayed pulse probed the exploding structure. The ejected ions were measured by ion time of flight spectrometry, and the particle sizes were measured by coherent diffractive imaging. The results show that sucrose particles of 560-1000 nm diameter retain their size for about 500 fs following the first exposure. Significant sample expansion happens between 500 fs and 1 ps. We present simulations to support these observations.
23.	Ribbeck-Busch, Kathrin, et al. (författare) A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila. 2005 Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 7:11 Tidskriftsartikel (refereegranskat)abstract In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.
24.	Valegård, Karin, et al. (författare) Structure of Rubisco from Arabidopsis thaliana in complex with 2-carboxyarabinitol-1,5-bisphosphate 2018 Ingår i: Acta Crystallographica Section D. - 2059-7983. ; 74:1, s. 1-9 Tidskriftsartikel (refereegranskat)abstract The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Arabidopsis thaliana is reported at 1.5 Å resolution. In light of the importance of A. thaliana as a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of an A. thaliana Rubisco crystal structure. A. thaliana Rubisco is an L8S8 hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits. A. thaliana produces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot of A. thaliana Rubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate. A. thaliana Rubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understand A. thaliana Rubisco and the potential catalytic differences that could be conferred by alternative A. thaliana Rubisco small-subunit isoforms.
25.	van der Schot, Gijs, et al. (författare) Imaging single cells in a beam of live cyanobacteria with an X-ray laser 2015 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6 Tidskriftsartikel (refereegranskat)abstract There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.

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