117 research outputs found
Heterologous expression, purification and structural features of native Dictyostelium discoideum dye-decolorizing peroxidase bound to a natively incorporated heme
The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis
Deliverable D4.4 Simulated coherent scattering data from plasma and non–plasma samples
Deliverable D4.4 of work package 4 (SIMEX) in EUCALL
Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging
The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample—a budding yeast cell (Saccharomyces cerevisiae)—using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution
Online dynamic flat-field correction for MHz Microscopy data at European XFEL
The X-ray microscopy technique at the European X-ray free-electron laser
(EuXFEL), operating at a MHz repetition rate, provides superior contrast and
spatial-temporal resolution compared to typical microscopy techniques at other
X-ray sources. In both online visualization and offline data analysis for
microscopy experiments, baseline normalization is essential for further
processing steps such as phase retrieval and modal decomposition. In addition,
access to normalized projections during data acquisition can play an important
role in decision-making and improve the quality of the data. However, the
stochastic nature of XFEL sources hinders the use of existing flat-flied
normalization methods during MHz X-ray microscopy experiments. Here, we present
an online dynamic flat-field correction method based on principal component
analysis of dynamically evolving flat-field images. The method is used for the
normalization of individual X-ray projections and has been implemented as an
online analysis tool at the Single Particles, Clusters, and Biomolecules and
Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.Comment: 14 pages, 7 figure
Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.11Ysciescopu
Ultrasound cavitation and exfoliation dynamics of 2D materials re-vealed in operando by X-ray free electron laser megahertz imaging
Ultrasonic liquid phase exfoliation is a promising method for the production
of two-dimensional (2D) layered materials. A large number of studies have been
made in investigating the underlying ultrasound exfoliation mechanisms.
However, due to the experimental challenges for capturing the highly transient
and dynamic phenomena in real-time at sub-microsecond time and micrometer
length scales simultaneously, most theories reported to date still remain
elusive. Here, using the ultra-short X-ray Free Electron Laser pulses (~25ps)
with a unique pulse train structure, we applied MHz X-ray Microscopy and
machine-learning technique to reveal unambiguously the full cycles of the
ultrasound cavitation and graphite layer exfoliation dynamics with
sub-microsecond and micrometer resolution. Cyclic fatigue shock wave impacts
produced by ultrasound cloud implosion were identified as the dominant
mechanism to deflect and exfoliate graphite layers mechanically. For the
graphite flakes, exfoliation rate as high as ~5 angstroms per shock wave impact
was observed. For the HOPG graphite, the highest exfoliation rate was ~0.15
angstroms per impact. These new findings are scientifically and technologically
important for developing industrial upscaling strategies for ultrasonic
exfoliation of 2D materials
Zu den Wurzeln der Modernen Architektur, Teil I
Modern emerging technologies, such as additive manufacturing, bioprinting, and new material production, require novel metrology tools to probe fundamental high-speed dynamics happening in such systems. Here we demonstrate the application of the megahertz (MHz) European X-ray Free-Electron Laser (EuXFEL) to image the fast stochastic processes induced by a laser on water-filled capillaries with micrometer-scale spatial resolution. The EuXFEL provides superior contrast and spatial resolution compared to equivalent state-of-the-art synchrotron experiments. This work opens up new possibilities for the characterization of MHz stochastic processes on the nanosecond to microsecond time scales with object velocities up to a few kilometers per second using XFEL sources
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