Collecting back-scattered X-rays from vacuum windows using a pinhole X-ray camera provides an efficient and reliable method of measuring the beam shape and position of the white synchrotron beam. In this paper, measurements are presented that were conducted at ESRF beamline ID6 which uses an in-vacuum cryogenically cooled permanent-magnet undulator (CPMU18) and a traditional U32 undulator as its radiation sources, allowing tests to be performed at very high power density levels that were adjusted by changing the gap of the undulators. These measurements show that it is possible to record beam shape and beam position using a simple geometry without having to place any further items in the beam path. With this simple test setup it was possible to record the beam position with a root-mean-square noise figure of 150 nm.

Gluconate 5-dehydrogenase (Ga5DH; EC 1.1.1.69) from Lentibacter algarum (LaGa5DH) was recombinantly expressed in Escherichia coli and purified to homogeneity. The protein was crystallized and the crystal structure was solved at 2.1 Å resolution. The crystal belonged to the monoclinic system, with space group P1 and unit-cell parameters a = 55.42, b = 55.48, c = 79.16 Å, α = 100.51, β = 105.66, γ = 97.99°. The structure revealed LaGaDH to be a tetramer, with each subunit consisting of six α-helices and three antiparallel β-hairpins. LaGa5DH has high structural similarity to other Ga5DH proteins, demonstrating that this enzyme is highly conserved.

Sample preparation within single-particle cryo-electron microscopy can still be a significant bottleneck, with issues in reproducibility, ice quality and sample loss. New approaches have recently been reported that use spraying or pin printing instead of the traditional blotting approach. Here, experience in the use of different nozzle designs and spraying regimes is reported together with their influence on the resulting spray and grid quality.

The title compound, C24H18FNO3, crystallizes in the monoclinic centrosymmetric space group P21/n and its mol­ecular conformation is stabilized via C—H⋯O intra­molecular inter­actions. The supra­molecular network mainly comprises C—H⋯O, C—H⋯F and C—H⋯π inter­actions, which contribute towards the formation of the crystal structure. The different inter­molecular inter­actions have been further analysed via Hirshfeld surface analysis and fingerprint plots.

The title compound, C19H16ClNO3S, consists of chloro­phenyl methyl­idene and di­hydro­benzo­thia­zine units linked to an acetate moiety, where the thia­zine ring adopts a screw-boat conformation. In the crystal, two sets of weak C—HPh⋯ODbt (Ph = phenyl and Dbt = di­hydro­benzo­thia­zine) hydrogen bonds form layers of mol­ecules parallel to the bc plane. The layers stack along the a-axis direction with inter­calation of the ester chains. The crystal studied was a two component twin with a refined BASF of 0.34961 (5). The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions to the crystal packing are from H⋯H (37.5%), H⋯C/C⋯H (24.6%) and H⋯O/O⋯H (16.7%) inter­actions. Hydrogen-bonding and van der Waals inter­actions are the dominant inter­actions in the crystal packing. Computational chemistry indicates that in the crystal, C—HPh⋯ODbt hydrogen bond energies are 38.3 and 30.3 kJ mol−1. Density functional theory (DFT) optimized structures at the B3LYP/ 6–311 G(d,p) level are compared with the experimentally determined mol­ecular structure in the solid state. The HOMO–LUMO behaviour was elucidated to determine the energy gap. Moreover, the anti­bacterial activity of the title compound has been evaluated against gram-positive and gram-negative bacteria.

Efficient infiltration of a mesoporous titania matrix with conducting organic polymers or small molecules is one key challenge to overcome for hybrid photovoltaic devices. A quantitative analysis of the backfilling efficiency with time-of-flight grazing incidence small-angle neutron scattering (ToF-GISANS) and scanning electron microscopy (SEM) measurements is presented. Differences in the morphology due to the backfilling of mesoporous titania thin films are compared for the macromolecule poly[4,8-bis­(5-(2-ethyl­hexyl)­thio­phen-2-yl)benzo[1,2-b;4,5-b']di­thio­phene-2,6-diyl-alt-(4-(2-ethyl­hexyl)-3-fluoro­thieno[3,4-b]thio­phene-)-2-carboxyl­ate-2-6-diyl)] (PTB7-Th) and the heavy-element containing small molecule 2-pinacol­boronate-3-phenyl­phen­anthro[9,10-b]telluro­phene (PhenTe-BPinPh). Hence, a 1.7 times higher backfilling efficiency of almost 70% is achieved for the small molecule PhenTe-BPinPh compared with the polymer PTB7-Th despite sharing the same volumetric mass density. The precise characterization of structural changes due to backfilling reveals that the volumetric density of backfilled materials plays a minor role in obtaining good backfilling efficiencies and interfaces with large surface contact.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air–water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

Anaerobic ammonium-oxidizing (anammox) bacteria play a key role in the global nitrogen cycle and in nitrogenous wastewater treatment. The anammox bacteria ultrastructure is unique and distinctly different from that of other prokaryotic cells. The morphological structure of an organism is related to its function; however, research on the ultrastructure of intact anammox bacteria is lacking. In this study, in situ three-dimensional nondestructive ultrastructure imaging of a whole anammox cell was performed using synchrotron soft X-ray tomography (SXT) and the total variation-based simultaneous algebraic reconstruction technique (TV-SART). Statistical and quantitative analyses of the intact anammox bacteria were performed. High soft X-ray absorption composition inside anammoxosome was detected and verified to be relevant to iron-binding protein. On this basis, the shape adaptation of the anammox bacteria response to iron was explored.

A historical tool for crystallographic analysis is provided by the Hilton net, which can be used for manually surveying the crystal lattice as it is manifested by the Kikuchi bands in a gnomonic projection. For a quantitative analysis using the Hilton net, the projection centre as the relative position of the signal source with respect to the detector plane needs to be known. Interplanar angles are accessible with a precision and accuracy which is estimated to be ≤0.3°. Angles between any directions, e.g. zone axes, are directly readable. Finally, for the rare case of an unknown projection-centre position, its determination is demonstrated by adapting an old approach developed for photogrammetric applications. It requires the indexing of four zone axes [uvw]i in a backscattered Kikuchi diffraction pattern of a known phase collected under comparable geometric conditions.

The sin2ψ and cosα methods are compared via diffracting-grain identification from electron backscatter diffraction maps. Artificial textures created by the X-ray diffraction measurements are plotted and X-ray elastic constants of the diffracting-grain sets are computed.

The crystal structure of gluconate 5-dehydrogenase from Lentibacter algarum is reported. It has high structural similarity to other gluconate 5-dehydrogenase proteins, demonstrating that this enzyme is highly conserved.

The true identity of the protein found in the crystals reported by Bondoc et al. [(2019), Acta Cryst. F75, 646–651] is given.

Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to α-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and α-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 Å resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities.

A new telescope has begun to virtually explore the solar system outback, and already is scoring discoveries.

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A new bacterial genome sequence could help researchers solve a mystery as to how microorganisms produce a highly toxic form of mercury.

The post New bacteria genome may help solve mystery of how methylmercury is made appeared first on Smithsonian Insider.

Slithering in at 48 feet long and weighing an estimated one-and-a-half tons, the largest snake the world has ever seen is being brought back to […]

The post Largest snake the world has ever seen is being brought back to life by Smithsonian Channel appeared first on Smithsonian Insider.

Paddling the remote oxbow lakes and bayous of the White River National Wildlife Refuge in Arkansas, the team of scientists was seeking proof of a […]

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The Republic of Panama’s proposal to implement four Traffic Separation Schemes for commercial vessels entering and exiting the Panama Canal and ports was approved unanimously […]

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What crunchy food did Americans spend $5.5 billion on last year—with sales that spiked before snow and ice storms? If you guessed birdseed then you […]

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A new study of microorganisms living on the skin and in the intestines of North America vultures (black and turkey vultures) has turned up a […]

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The National Zoo’s red panda habitat on Asia Trail reopened to the public Tuesday, May 10. Tusa and Asa explored their new home which has […]

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Crustaceans that thrive in the vastness of the open ocean have no place to hide from their predators. Consequently, many creatures that live at depths […]

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In the early 20th century, Canada geese were considered endangered in the U.S. So in the 1950s and 1960s, birds from the Midwest were released […]

The post Study of bacteria inside guts of wild Canada geese shows greater danger than earlier studies exposed appeared first on Smithsonian Insider.

Little to nothing is known about how and where a small European songbird called the bluethroat spends much of the year. Now, Smithsonian scientists have […]

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Pullulanase (EC 3.2.1.41) is a well known starch-debranching enzyme that catalyzes the cleavage of α-1,6-glycosidic linkages in α-glucans such as starch and pullulan. Crystal structures of a type I pullulanase from Paenibacillus barengoltzii (PbPulA) and of PbPulA in complex with maltopentaose (G5), maltohexaose (G6)/α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD) were determined in order to better understand substrate binding to this enzyme. PbPulA belongs to glycoside hydrolase (GH) family 13 subfamily 14 and is composed of three domains (CBM48, A and C). Three carbohydrate-binding sites identified in PbPulA were located in CBM48, near the active site and in domain C, respectively. The binding site in CBM48 was specific for β-CD, while that in domain C has not been reported for other pullulanases. The domain C binding site had higher affinity for α-CD than for G6; a small motif (FGGEH) seemed to be one of the major determinants for carbohydrate binding in this domain. Structure-based mutations of several surface-exposed aromatic residues in CBM48 and domain C had a debilitating effect on the activity of the enzyme. These results suggest that both CBM48 and domain C play a role in binding substrates. The crystal forms described contribute to the understanding of pullulanase domain–carbohydrate interactions.

Julian Chollet, Alexander Dünkler, Anne Bäuerle, Laura Vivero-Pol, Medhanie A. Mulaw, Thomas Gronemeyer, and Nils Johnsson

Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of the septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. Hereby, the septin-rods follow the high concentration of Cdc42_GTP that is generated by the focused localization of its GEF Cdc24. We show that shortly before budding Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 reducing the affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 is found at the cortex of the bud and directs its further outgrowth from this position.

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Current plans to protect the Chesapeake Bay include planting trees along hundreds of miles of streams that empty into the Bay. This study provides realistic limits for how much these buffers might further reduce nitrate pollution, and it helps identify where buffer restoration can offer the greatest additional nitrate removal.

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Recently a team of scientists working at the Smithsonian’s Tropical Research Institute in Panama, decided to try and learn for the first time just which body parts the gliding ant C. atratus uses to steer as they glide.

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Nutmeg-loving toucans wearing GPS transmitters recently helped a team of scientists at the Smithsonian Tropical Research Institute in Panama address an age-old problem in plant ecology: accurately estimating seed dispersal.

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Christopher R. Mahone
Apr 8, 2020; 133:jcs237057-jcs237057
CELL SCIENCE AT A GLANCE

Full Text:

The molecular mechanism used by many bacteria to kill neighboring cells has redundancy built into its genetic makeup, which could allow for the mechanism to be expressed in different environments, say researchers at Penn State and the University of Wisconsin-Madison. Their new study provides insights into the molecular mechanisms of competition among bacteria. "Many organisms, including humans, acquire bacteria from their environment," said Tim Miyashiro, a biochemist and molecular biologist at Penn State and the leader of the research team. "These bacteria can contribute to functions within the host organism, like how our gut bacteria help us digest food. We're interested in the interactions among bacteria cells, and between bacteria and their hosts, to better understand these mutually beneficial symbiotic relationships." Cells of the bioluminescent bacteria Vibrio fisheri take up residence in the light organ of newly hatched bobtail squid. At night, the bacteria produce a blue glow that researchers believe obscures a squid's silhouette and helps protect it from predators. The light organ has pockets, or crypts, in the squid's skin that provide nutrients and a safe environment for the bacteria. "When the squid hatches, it doesn't yet have any bacteria in its light organ," said Miyashiro. "But bacteria in the environment quickly colonize the squid's light organ." Some of these different bacteria strains can coexist, but others can't. "Microbial symbioses are essentially universal in animals, and are crucial to the health and development of both partners," says Irwin Forseth, a program director in the National Science Foundation's Division of Integrative Organismal Systems, which funded the research. "The results from this study highlight the role small genetic changes can play in microbe interactions. Increased understanding will allow us to better predict organisms' performance in changing environments."

Image credit: Andrew Cecere

Americas progress in arresting its obesity epidemic has been too slow, and the condition continues to erode productivity and cause millions to suffer from potentially debilitating and deadly chronic illnesses.