The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportion­ation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

The refinement of biomolecular crystallographic models relies on geometric restraints to help to address the paucity of experimental data typical in these experiments. Limitations in these restraints can degrade the quality of the resulting atomic models. Here, an integration of the full all-atom Amber molecular-dynamics force field into Phenix crystallographic refinement is presented, which enables more complete modeling of biomolecular chemistry. The advantages of the force field include a carefully derived set of torsion-angle potentials, an extensive and flexible set of atom types, Lennard–Jones treatment of nonbonded interactions and a full treatment of crystalline electrostatics. The new combined method was tested against conventional geometry restraints for over 22 000 protein structures. Structures refined with the new method show substantially improved model quality. On average, Ramachandran and rotamer scores are somewhat better, clashscores and MolProbity scores are significantly improved, and the modeling of electrostatics leads to structures that exhibit more, and more correct, hydrogen bonds than those refined using traditional geometry restraints. In general it is found that model improvements are greatest at lower resolutions, prompting plans to add the Amber target function to real-space refinement for use in electron cryo-microscopy. This work opens the door to the future development of more advanced applications such as Amber-based ensemble refinement, quantum-mechanical representation of active sites and improved geometric restraints for simulated annealing.

Corrections are published for the article by Caldararu et al. [(2019), Acta Cryst. D75, 368–380].

Noncrystallographic symmetry (NCS) averaging following molecular-replacement phasing is generally the major technique used to solve a structure with several molecules in one asymmetric unit, such as a spherical icosahedral viral particle. As an alternative method to NCS averaging, a new approach to optimize or to refine the electron density directly under NCS constraints is proposed. This method has the same effect as the conventional NCS-averaging method but does not include the process of Fourier synthesis to generate the electron density from amplitudes and the corresponding phases. It has great merit for the solution of structures with limited data that are either twinned or incomplete at low resolution. This method was applied to the case of the T = 1 shell-domain subviral particle of Penaeus vannamei nodavirus with data affected by twinning using the REFMAC5 refinement software.

Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X-ray absorption edges, which shift to higher energy for progressively more positive valence numbers. Indeed, X-ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X-ray sources at synchrotron facilities with energy resolutions in the range 2–3 × 10−4 (ΔE/E). This paper tests whether X-ray free-electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10−3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength-dependent anomalous scattering factors contributed by two differently charged iron centers in the 110-amino-acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X-ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.

The performance of automated protein model building usually decreases with resolution, mainly owing to the lower information content of the experimental data. This calls for a more elaborate use of the available structural information about macromolecules. Here, a new method is presented that uses structural homologues to improve the quality of protein models automatically constructed using ARP/wARP. The method uses local structural similarity between deposited models and the model being built, and results in longer main-chain fragments that in turn can be more reliably docked to the protein sequence. The application of the homology-based model extension method to the example of a CFA synthase at 2.7 Å resolution resulted in a more complete model with almost all of the residues correctly built and docked to the sequence. The method was also evaluated on 1493 molecular-replacement solutions at a resolution of 4.0 Å and better that were submitted to the ARP/wARP web service for model building. A significant improvement in the completeness and sequence coverage of the built models has been observed.

The first experimental results from a new transmissive diagnostic instrument for synchrotron X-ray beamlines are presented. The instrument utilizes a single-crystal chemical-vapour-deposition diamond plate as the detector material, with graphitic wires embedded within the bulk diamond acting as electrodes. The resulting instrument is an all-carbon transmissive X-ray imaging detector. Within the instrument's transmissive aperture there is no surface metallization that could absorb X-rays, and no surface structures that could be damaged by exposure to synchrotron X-ray beams. The graphitic electrodes are fabricated in situ within the bulk diamond using a laser-writing technique. Two separate arrays of parallel graphitic wires are fabricated, running parallel to the diamond surface and perpendicular to each other, at two different depths within the diamond. One array of wires has a modulated bias voltage applied; the perpendicular array is a series of readout electrodes. X-rays passing through the detector generate charge carriers within the bulk diamond through photoionization, and these charge carriers travel to the nearest readout electrode under the influence of the modulated electrical bias. Each of the crossing points between perpendicular wires acts as an individual pixel. The simultaneous read-out of all pixels is achieved using a lock-in technique. The parallel wires within each array are separated by 50 µm, determining the pixel pitch. Readout is obtained at 100 Hz, and the resolution of the X-ray beam position measurement is 600 nm for a 180 µm size beam.

The Python programming language, combined with the numerical computing library NumPy and the scientific computing library SciPy, has become the de facto standard for scientific computing in a variety of fields. This popularity is mainly due to the ease with which a Python program can be written and executed (easy syntax, dynamical typing, no compilation etc.), coupled with the existence of a large number of specialized third-party libraries that aim to lift all the limitations of the raw Python language. NumPy introduces vector programming, improving execution speeds, whereas SciPy brings a wealth of highly optimized and reliable scientific functions. There are cases, however, where vector programming alone is not sufficient to reach optimal performance. This issue is addressed with dedicated compilers that aim to translate Python code into native and statically typed code with support for the multi-core architectures of modern processors. In the present article it is shown how these approaches can be efficiently used to tackle different problems, with increasing complexity, that are relevant to crystallography: the 2D Laue function, scattering from a strained 2D crystal, scattering from 3D nanocrystals and, finally, diffraction from films and multilayers. For each case, detailed implementations and explanations of the functioning of the algorithms are provided. Different Python compilers (namely NumExpr, Numba, Pythran and Cython) are used to improve performance and are benchmarked against state-of-the-art NumPy implementations. All examples are also provided as commented and didactic Python (Jupyter) notebooks that can be used as starting points for crystallographers curious to enter the Python ecosystem or wishing to accelerate their existing codes.

The section of the Bilbao Crystallographic Server (http://www.cryst.ehu.es) dedicated to subperiodic groups includes a new tool called LSITESYM for the study of materials with layer and multilayer symmetry. This new program, based on the site-symmetry approach, establishes the symmetry relations between localized and extended crystal states using representations of layer groups. The efficiency and utility of the program LSITESYM is demonstrated by illustrative examples, which include the analysis of phonon symmetry in Aurivillius compounds and in van der Waals layered crystals MoS2 and WS2.

DatView is a new graphical user interface (GUI) for plotting parameters to explore correlations, identify outliers and export subsets of data. It was designed to simplify and expedite analysis of very large unmerged serial femtosecond crystallography (SFX) data sets composed of indexing results from hundreds of thousands of microcrystal diffraction patterns. However, DatView works with any tabulated data, offering its functionality to many applications outside serial crystallography. In DatView's user-friendly GUI, selections are drawn onto plots and synchronized across all other plots, so correlations between multiple parameters in large multi-parameter data sets can be rapidly identified. It also includes an item viewer for displaying images in the current selection alongside the associated metadata. For serial crystallography data processed by indexamajig from CrystFEL [White, Kirian, Martin, Aquila, Nass, Barty & Chapman (2012). J. Appl. Cryst. 45, 335–341], DatView generates a table of parameters and metadata from stream files and, optionally, the associated HDF5 files. By combining the functionality of several commonly needed tools for SFX in a single GUI that operates on tabulated data, the time needed to load and calculate statistics from large data sets is reduced. This paper describes how DatView facilitates (i) efficient feedback during data collection by examining trends in time, sample position or any parameter, (ii) determination of optimal indexing and integration parameters via the comparison mode, (iii) identification of systematic errors in unmerged SFX data sets, and (iv) sorting and highly flexible data filtering (plot selections, Boolean filters and more), including direct export of subset CrystFEL stream files for further processing.

Bragg intensities can be used to analyse crystal size distributions in a method called FXD-CSD, which is based on the fast measurement of many Bragg spots using two-dimensional detectors. This work presents the Python-based software and its graphical user interface FXD-CSD-GUI. The GUI enables user-friendly data handling and processing and provides both graphical and numerical crystal size distribution results.

Crystallographic textures, as they develop for example during cold forming, can have a significant influence on the mechanical properties of metals, such as plastic anisotropy. Textures are typically characterized by a non-uniform distribution of crystallographic orientations that can be measured by diffraction experiments like electron backscatter diffraction (EBSD). Such experimental data usually contain a large number of data points, which must be significantly reduced to be used for numerical modeling. However, the challenge in such data reduction is to preserve the important characteristics of the experimental data, while reducing the volume and preserving the computational efficiency of the numerical model. For example, in micromechanical modeling, representative volume elements (RVEs) of the real microstructure are generated and the mechanical properties of these RVEs are studied by the crystal plasticity finite element method. In this work, a new method is developed for extracting a reduced set of orientations from EBSD data containing a large number of orientations. This approach is based on the established integer approximation method and it minimizes its shortcomings. Furthermore, the L1 norm is applied as an error function; this is commonly used in texture analysis for quantitative assessment of the degree of approximation and can be used to control the convergence behavior. The method is tested on four experimental data sets to demonstrate its capabilities. This new method for the purposeful reduction of a set of orientations into equally weighted orientations is not only suitable for numerical simulation but also shows improvement in results in comparison with other available methods.

Over the past decade, ptychography has been proven to be a robust tool for non-destructive high-resolution quantitative electron, X-ray and optical microscopy. It allows for quantitative reconstruction of the specimen's transmissivity, as well as recovery of the illuminating wavefront. Additionally, various algorithms have been developed to account for systematic errors and improved convergence. With fast ptychographic microscopes and more advanced algorithms, both the complexity of the reconstruction task and the data volume increase significantly. PtychoShelves is a software package which combines high-level modularity for easy and fast changes to the data-processing pipeline, and high-performance computing on CPUs and GPUs.

Book review

EDDIDAT is a program that provides a graphical user interface (GUI) for the evaluation of energy-dispersive X-ray diffraction data with the focus on the depth-resolved residual stress analysis.

The synthesis, characterization and study of structures from a series of bi­phenyls substituted at positions 3, 3', 4 and 4' with groups connected to the bi­phenyl core through oxygen atoms are presented here. The molecular conformation is extensively studied both in the solid as well as in the liquid state, and the effect of different actors (such as packing and chain length) on the torsion angle between aromatic rings is analyzed.

Crystal structure and microscopic optical properties of anthracene derivative compounds have been investigated by single-crystal synchrotron X-ray diffraction, laser confocal microscopy and fluorescence lifetime imaging microscopy.

Insights were obtained into the structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway for the biosynthesis of lysine.

This article reports conformational polymorphisms of the EF-hand protein MCFD2 which is involved in glycoprotein transport..

There are several hypotheses about how amphibian chytrid has spread around the world, but the trade in amphibians for food, bait, pets and laboratory animals has been identified as the most likely mode of spread

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The rescue pods will be part of the project’s Amphibian Rescue Center at Summit Municipal Park, which will also include a lab with a quarantine facility.

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By compiling and comparing long-term data from stranding records and visual sighting records, both taken from nearly every ocean basin in the world, Pyenson verified that stranding records “faithfully reflect the number of species and the relative abundance” found in live surveys.

The post Stranding records are faithful reflection of live whale and dolphin populations, new study reveals appeared first on Smithsonian Insider.

Any adventurer, hiker or backyard naturalist with a camera can help scientists survey and hopefully save the world’s amphibians thanks to a new social networking site that links “citizen scientists” with researchers tracking the decline of amphibians around the globe.

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Smithsonian scientists have confirmed that chytridiomycosis, a rapidly spreading amphibian disease, has reached a site near Panama’s Darien region. This was the last area in the entire mountainous neotropics to be free of the disease. This is troubling news for the Panama Amphibian Rescue and Conservation Project, a consortium of nine U.S. and Panamanian institutions that aims to rescue 20 species of frogs in imminent danger of extinction.

The post Deadly amphibian disease detected in the last disease-free region of Central America appeared first on Smithsonian Insider.

Reptiles that live near and feed upon amphibians in the tropics may be spreading the deadly amphibian disease Chytridiomycosis (caused by the chytrid fungus Batrachochytrium dedrobatidis), holding and transporting reservoirs of the fungus on their skin.

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Justin Touchon, post-doctoral fellow at the Smithsonian Tropical Research Institute, discovered that climate change in Panama may be altering frogs’ course of evolution.

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Bright orange with a distinctive call the Panamanian poison dart frog Andinobates geminisae lives in only a small area of the Caribbean Coast of Panama. […]

The post In modern amphibian “ark,” new species added with due diligence appeared first on Smithsonian Insider.

Tiny and transparent the marine crustacean Paraphronima gracilis sees the world through two large eyes that envelope its head like a high-tech space helmet. Now, […]

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Smithsonian scientists and colleagues have discovered a new genus and species of river dolphin that has long been extinct. They made the discovery after carefully […]

The post Fossil Specimen Reveals a New Species of Ancient River Dolphin appeared first on Smithsonian Insider.

A fossil that has been in the collection of the Smithsonian’s National Museum of Natural History since it was discovered in 1951 is today helping […]

The post New Species of Extinct River Dolphin Discovered in Smithsonian Collection appeared first on Smithsonian Insider.

Marine mammologist Matthew Leslie aims his crossbow from the bow of a moving boat at the dolphins riding the breaking waves below. A dolphin will […]

The post Some dolphins cross the Pacific more easily than others. Why that matters for protecting them appeared first on Smithsonian Insider.

Now, for the first time, Charles Darwin's life is portrayed pictorially in an illustrated biography in graphic novel-style for all ages to enjoy.

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In the first volume of the Secret Smithsonian Adventures graphic-novel series from Smithsonian Books, The Wrong Wrights, four middle-school kids visit the Smithsonian’s National Air […]

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Scott J. Neal, Qingxiang Zhou, and Francesca Pignoni

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.

Bipasha Dey and Richa Rikhy

Cell shape morphogenesis from spherical to polygonal occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. In this study, we assess the relative contribution of DE-cadherin and Myosin II at the furrow for polygonal shape transition. We show that polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 µm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of polygonal shape transition. Our studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.

Aparna Sherlekar, Gayatri Mundhe, Prachi Richa, Bipasha Dey, Swati Sharma, and Richa Rikhy

Branched actin networks driven by Arp2/3 collaborate with actomyosin filaments in processes such as cell migration. The syncytial Drosophila blastoderm embryo also shows expansion of apical caps by Arp2/3 driven actin polymerization in interphase and buckling at contact edges by MyosinII to form furrows in metaphase. Here we study the role of Syndapin (Synd), an F-BAR domain containing protein in apical cap remodelling prior to furrow extension. synd depletion showed larger apical caps. STED super-resolution and TIRF microscopy showed long apical actin protrusions in caps in interphase and short protrusions in metaphase in control embryos. synd depletion led to sustained long protrusions even in metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodelling. MyosinII levels were decreased in synd mutants and MyosinII mutant embryos have been previously reported to have expanded caps. We propose that Syndapin function limits branching activity during cap expansion and affects MyosinII distribution in order to shift actin remodeling from apical cap expansion to favor lateral furrow extension.

Join National Zoo Conservation Biologist Brian Gratwicke in his work to save Panama's amphibians from extinction. You can also catch up on his dispatches from the field on the Panamanian Amphibian Rescue and Conservation Project's blog: http://amphibianrescue.org/

The post Meet our Scientist: Brian Gratwicke, Amphibian Avenger appeared first on Smithsonian Insider.

The Smithsonian Conservation Biology Institute and the Smithsonian Tropical Research Institute are working together as part of the Panama Amphibian Rescue and Conservation Project have […]

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The unit-cell reduction described by Selling and used by Delone (whose early publications were under the spelling Delaunay) is explained in a simple form. The transformations needed to implement the reduction are listed. The simplicity of this reduction contrasts with the complexity of Niggli reduction.

An intuitive method is presented for detecting pseudosymmetries in Z' > 1 cases as a complement to well-proven strategies already available in the literature. It is based in the simple idea that the mid-points between equivalent atoms in symmetrically related mol­ecules are disposed according to simple well-known patterns, which are easily recognizable by optical inspection. A number of Z' = 4 cases in the literature are analyzed, which allows some of the potentialities of the method to be revealed.