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

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

First operation of the JUNGFRAU detector in 16-memory cell mode at European XFEL

The JUNGFRAU detector is a well-established hybrid pixel detector developed at the Paul Scherrer Institut (PSI) designed for free-electron laser (FEL) applications. JUNGFRAU features a charge-integrating dynamic gain switching architecture, with three different gain stages and 75 μm pixel pitch. It is widely used at the European X-ray Free-Electron Laser (EuXFEL), a facility which produces high brilliance X-ray pulses at MHz repetition rate in the form of bursts repeating at 10 Hz. In nominal configuration, the detector utilizes only a single memory cell and supports data acquisition up to 2 kHz. This constrains the operation of the detector to a 10 Hz frame rate when combined with the pulsed train structure of the EuXFEL. When configured in so-called burst mode, the JUNGFRAU detector can acquire a series of images into sixteen memory cells at a maximum rate of around 150 kHz. This acquisition scheme is better suited for the time structure of the X-rays as well as the pump laser pulses at the EuXFEL. To ensure confidence in the use of the burst mode at EuXFEL, a wide range of measurements have been performed to characterize the detector, especially to validate the detector alibration procedures. In particular, by analyzing the detector response to varying photon intensity (so called ‘intensity scan’), special attention was given to the characterization of the transitions between gain stages. The detector was operated in both dynamic gain switching and fixed gain modes. Results of these measurements indicate difficulties in the characterization of the detector dynamic gain switching response while operated in burst mode, while no major issues have been found with fixed gain operation. Based on this outcome, fixed gain operation mode with all the memory cells was used during two experiments at EuXFEL, namely in serial femtosecond protein crystallography and Kossel lines measurements. The positive outcome of these two experiments validates the good results previously obtained, and opens the possibility for a wider usage of the detector in burst operation mode, although compromises are needed on the dynamic range

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

18 pags, 11 figs, 5 tabsHere, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.This work was supported by the National Science Foundation Science and Technology Center 'BioXFEL' through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DESC0002164 (AO, algorithm design and development) and by the National Science Foundation under contract Nos. 1551489 (AO, underlying analytical models) and DBI-2029533 (AO, functional conformations). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 to JLO. The work was also supported by funds from the National Institutes of Health grant R01 GM117342-0404. Funding and support are also acknowledged from the National Institutes of Health grant R01 GM095583, from the Biodesign Center for Applied Structural Discovery at ASU, from National Science Foundation award No. 1565180 and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. KAZ was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). This work was also supported by the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. CFEL is supported by the Gottfried Wilhelm Leibniz Program of the DFG, the 'X-probe' project funded by the European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement 637295, the European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)', ERC-2013-SyG 609920, and the Human Frontiers Science Program grant RGP0010 2017. This work is also supported by the AXSIS project funded by the European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 609920.Peer reviewe

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

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods

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 H$_2$O$_2$-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

3D atomic structure from a single X-ray free electron laser pulse

Abstract X-ray Free Electron Lasers (XFEL) are cutting-edge pulsed x-ray sources, whose extraordinary pulse parameters promise to unlock unique applications. Several new methods have been developed at XFELs; however, no methods are known, which allow ab initio atomic level structure determination using only a single XFEL pulse. Here, we present experimental results, demonstrating the determination of the 3D atomic structure from data obtained during a single 25 fs XFEL pulse. Parallel measurement of hundreds of Bragg reflections was done by collecting Kossel line patterns of GaAs and GaP. To the best of our knowledge with these measurements, we reached the ultimate temporal limit of the x-ray structure solution possible today. These measurements open the way for obtaining crystalline structures during non-repeatable fast processes, such as structural transformations. For example, the atomic structure of matter at extremely non-ambient conditions or transient structures formed in irreversible physical, chemical, or biological processes may be captured in a single shot measurement during the transformation. It would also facilitate time resolved pump-probe structural studies making them significantly shorter than traditional serial crystallography

EXtra-Xwiz: A Tool to Streamline Serial Femtosecond Crystallography Workflows at European XFEL

X-ray free electron lasers deliver photon pulses that are bright enough to observe diffraction from extremely small crystals at a time scale that outruns their destruction. As crystals are continuously replaced, this technique is termed serial femtosecond crystallography (SFX). Due to its high pulse repetition rate, the European XFEL enables the collection of rich and extensive data sets, which are suited to study various scientific problems, including ultra-fast processes. The enormous data rate, data complexity, and the nature of the pixelized multimodular area detectors at the European XFEL pose severe challenges to users. To streamline the analysis of the SFX data, we developed the semiautomated pipeline EXtra-Xwiz around the established CrystFEL program suite, thereby processing diffraction patterns on detector frames into structure factors. Here we present EXtra-Xwiz, and we introduce its architecture and use by means of a tutorial. Future plans for its development and expansion are also discussed

3D-printed sheet jet for stable megahertz liquid sample delivery at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) can probe chemical and biological reactions as they unfold with unprecedented spatial and temporal resolution. A principal challenge in this pursuit involves the delivery of samples to the X-ray interaction point in such a way that produces data of the highest possible quality and with maximal efficiency. This is hampered by intrinsic constraints posed by the light source and operation within a beamline environment. For liquid samples, the solution typically involves some form of high-speed liquid jet, capable of keeping up with the rate of X-ray pulses. However, conventional jets are not ideal because of radiation-induced explosions of the jet, as well as their cylindrical geometry combined with the X-ray pointing instability of many beamlines which causes the interaction volume to differ for every pulse. This complicates data analysis and contributes to measurement errors. An alternative geometry is a liquid sheet jet which, with its constant thickness over large areas, eliminates the problems related to X-ray pointing. Since liquid sheets can be made very thin, the radiation-induced explosion is reduced, boosting their stability. These are especially attractive for experiments which benefit from small interaction volumes such as fluctuation X-ray scattering and several types of spectroscopy. Although their use has increased for soft X-ray applications in recent years, there has not yet been wide-scale adoption at XFELs. Here, gas-accelerated liquid sheet jet sample injection is demonstrated at the European XFEL SPB/SFX nano focus beamline. Its performance relative to a conventional liquid jet is evaluated and superior performance across several key factors has been found. This includes a thickness profile ranging from hundreds of nanometres to 60 nm, a fourfold increase in background stability and favorable radiation-induced explosion dynamics at high repetition rates up to 1.13 MHz. Its minute thickness also suggests that ultrafast single-particle solution scattering is a possibility