86 research outputs found
A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source
Complementary techniques greatly aid the interpretation of macromolecule structures to yield functional information, and can also help to track radiation-induced changes. A new on-axis spectrometer being integrated into the macromolecular crystallography beamlines of the Swiss Light Source is presented
Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser.
Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5\u27-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1â
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resolution. This model, which was refined against a data set from âŒ750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100â
K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding
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
Development of crystal optics for Multi-Projection X-ray Imaging for synchrotron and XFEL sources
X-ray Multi-Projection Imaging (XMPI) is an emerging technology that allows
for the acquisition of millions of 3D images per second in samples opaque to
visible light. This breakthrough capability enables volumetric observation of
fast stochastic phenomena, which were inaccessible due to the lack of a
volumetric X-ray imaging probe with kHz to MHz repetition rate. These include
phenomena of industrial and societal relevance such as fractures in solids,
propagation of shock waves, laser-based 3D printing, or even fast processes in
the biological domain. Indeed, the speed of traditional tomography is limited
by the shear forces caused by rotation, to a maximum of 1000 Hz in
state-of-the-art tomography. Moreover, the shear forces can disturb the
phenomena in observation, in particular with soft samples or sensitive
phenomena such as fluid dynamics. XMPI is based on splitting an X-ray beam to
generate multiple simultaneous views of the sample, therefore eliminating the
need for rotation. The achievable performances depend on the characteristics of
the X-ray source, the detection system, and the X-ray optics used to generate
the multiple views. The increase in power density of the X-ray sources around
the world now enables 3D imaging with sampling speeds in the kilohertz range at
synchrotrons and megahertz range at X-ray Free-Electron Lasers (XFELs). Fast
detection systems are already available, and 2D MHz imaging was already
demonstrated at synchrotron and XFEL. In this work, we explore the properties
of X-ray splitter optics and XMPI schemes that are compatible with synchrotron
insertion devices and XFEL X-ray beams. We describe two possible schemes
designed to permit large samples and complex sample environments. Then, we
present experimental proof of the feasibility of MHz-rate XMPI at the European
XFEL.Comment: 47 pages, 17 figure
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
Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections
Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptinâs efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections
Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease
The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics
X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease
The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2
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