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Physical review applied 17(4), 044044 (2022). doi:10.1103/PhysRevApplied.17.044044, Photophoretic forces are induced when light causes a net momentum exchange between a particle and a surrounding gas. Such forces have been shown to be a robust means for trapping and guiding particles in air over long distances. Here, we apply the concept of an optical funnel for the delivery of bioparticles to the focus of an x-ray free-electron laser (XFEL) for femtosecond x-ray diffractive imaging. We provide the experimental demonstration of transversely compressing a high-speed beam of aerosolized viruses via photophoretic forces in a low-pressure gas environment. Relative temperature gradients induced on the viruses by the laser are estimated via particle-velocimetry measurements. The results demonstrate the potential for an optical funnel to improve particle-delivery efficiency in XFEL imaging and spectroscopy., Published by American Physical Society, College Park, Md. [u.a.]

Scientific data 3, 160064 -(2016). doi:10.1038/sdata.2016.64, 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 well-characterized 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 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström 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., Published by Nature Publ. Group, London

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s−1, which allows for the use of megahertz XFEL repetition rates. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering., There is a need for more robust sample delivery methods for serial crystallography. Here the authors present the design and characterization of ultracompact 3D microfluidic devices that can be printed, which require less sample, have a lower background signal and allow 3D mixing for time resolved experiments.

Tpp49Aa1 fromLysinibacillus sphaericusis a Toxin_10 family protein that – in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically againstCulex quinquefasciatusmosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 Å resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae fromAnopheles stephensi, Aedes albopictusandCulex tarsalis– substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained usingCulexcell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components.Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 Å.Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity.We expand the known target range to include three more mosquito species:Anopheles stephensi, Aedes albopictusandCulex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

High-speed liquid micro-jets are used to rapidly and repeatedly deliver protein microcrystals to focused and pulsed X-ray beams in the method of serial femtosecond crystallography. However, the current continuous flow of crystals is mismatched to the arrival of X-ray pulses, wasting vast amounts of an often rare and precious sample. Here, we introduce a method to address this problem by periodically trapping and releasing crystals in the liquid flow, creating locally concentrated crystal bunches, using an optical trap integrated in the microfluidic supply line. We experimentally demonstrate a 30-fold increase of particle concentration into 10 Hz bunches of 6.4 μm diameter polystyrene particles. Furthermore, using particle trajectory simulations, a comprehensive description of the optical bunching process and parameter space is presented. Adding this compact optofluidics device to existing injection systems would thereby dramatically reduce sample consumption and extend the application of serial crystallography to a greater range of protein crystal systems that cannot be produced in high abundance. Our approach is suitable for other microfluidic systems that require synchronous measurements of flowing objects.

We report on the use of multilayer Laue lenses to focus the intense X-ray Free Electron Laser (XFEL) beam at the European XFEL to a spot size of a few tens of nanometers. We present the procedure to align and characterize these lenses and discuss challenges working with the pulse trains from this unique x-ray source.

A large-scale screen to target SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, Mpro, is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to Mpro. High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site. Science, this issue p. 642, A repurposed drug-library screen reveals two allosteric drug binding sites of the 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 (Mpro), 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 Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

18 pags, 11 figs, 5 tabs, Here, 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.

Optica 8(1), 15 - 23 (2021). doi:10.1364/OPTICA.410851, Single particle imaging at x-ray free electron lasers (XFELs) has the potential to determine the structure and dynamics of single biomolecules at room temperature. Two major hurdles have prevented this potential from being reached, namely, the collection of sufficient high-quality diffraction patterns and robust computational purification to overcome structural heterogeneity. We report the breaking of both of these barriers using gold nanoparticle test samples, recording around 10 million diffraction patterns at the European XFEL and structurally and orientationally sorting the patterns to obtain better than 3-nm-resolution 3D reconstructions for each of four samples. With these new developments, integrating advancements in x-ray sources, fast-framing detectors, efficient sample delivery, and data analysis algorithms, we illuminate the path towards sub-nanometer biomolecular imaging. The methods developed here can also be extended to characterize ensembles that are inherently diverse to obtain their full structural landscape., Published by OSA, Washington, DC

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 two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. 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. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors.

We present a comparison between a numerical model and the experimental results on the generation of a first-order Bessel-Gaussian optical funnel for guiding of airborne microparticles.

Facilitating the very short and intense pulses from an X-ray laser for the purpose of imaging small bioparticles carries the potential for structure determination at atomic resolution without the need for crystallization. In this study, experimental strategies for this idea are explored based on data collected at the Linac Coherent Light Source from 40 nm virus particles injected into a hard X-ray beam., This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of ∼40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from ∼35 to ∼300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 × 1012 photons per µm2 per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.

The journal of physical chemistry letters 10(3), 441 - 446 (2019). doi:10.1021/acs.jpclett.8b03595, X-ray free electron lasers (XFELs) provide ultrashort intense X-ray pulses suitable to probe electron dynamics but can also induce a multitude of nonlinear excitation processes. These affect spectroscopic measurements and interpretation, particularly for upcoming brighter XFELs. Here we identify and discuss the limits to observing classical spectroscopy, where only one photon is absorbed per atom for a Mn$^{2+}$ in a light element (O, C, H) environment. X-ray emission spectroscopy (XES) with different incident photon energies, pulse intensities, and pulse durations is presented. A rate equation model based on sequential ionization and relaxation events is used to calculate populations of multiply ionized states during a single pulse and to explain the observed X-ray induced spectral lines shifts. This model provides easy estimation of spectral shifts, which is essential for experimental designs at XFELs and illustrates that shorter X-ray pulses will not overcome sequential ionization but can reduce electron cascade effects., Published by ACS, Washington, DC

Scientific Reports 7: Article number: 44628; published online: 16 March 2017; updated: 21 June 2017. In this Article, Henry N. Chapman is incorrectly listed as being affiliated with ‘Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA’ and an additional affiliation was omitted.

A numerical simulation infrastructure capable of calculating the flow of gas and the trajectories of particles through an aerodynamic lens injector is presented. The simulations increase the fundamental understanding and predict optimized injection geometries and parameters. Our simulation results were compared to previous reports and also validated against experimental data for 500 nm polystyrene spheres from an aerosol-beam-characterization setup. The simulations yielded a detailed understanding of the radial phase-space distribution and highlighted weaknesses of current aerosol injectors for single-particle diffractive imaging. With the aid of these simulations we developed new experimental implementations to overcome current limitations.

Scientific data 4, 170079 (2017). doi:10.1038/sdata.2017.79, Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single- particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency., Published by Macmillan, London

SPIE Nanoscience + Engineering, San Diego, California, 17 Aug 2016 - 21 Aug 2016 ; Proceedings of SPIE 9022, 99220K(2016). doi:10.1117/12.2239171, Optical trapping of light-absorbing particles in a gas environment is usually dominated by laser-induced thermal or photophoretic forces, which can be orders of magnitude higher than the force due to radiation pressure. Particle guiding with photophoretic forces over large distances in open air was recently realised by an optical pipeline, formed by a vortex laser beam of doughnut-like intensity profile, with a high-intensity ring of light that surrounds a dark core. We are adapting the optical pipeline concept for the purpose of guiding aerosolized particles into the intense focus of a x-ray free-electron laser (XFEL), in order to enable high-efficiency femtosecond x-ray coherent diffractive imaging (CDI). XFEL-based CDI allows single-shot nanometer-resolution imaging, and multi-shot Angstrom-resolution tomography in the case of reproducible nanoparticles, at a time resolution better than 10 femtoseconds. Remarkably, by imaging at timescales shorter than atomic motion, the crucial resolution-limiting effects of radiation damage may be overcome for radiation-sensitive targets such as viruses and biomolecules. Following on our previous work, we are developing an optical first-order Bessel-like beam with a variable-diameter hollow core and an axial-to-lateral aspect ratio up to ~2000, that can be used to guide particles with a spatial precision of less than a few µm over centimetre-long distances. We present the ways to control the beam divergence aiming to focus the stream of particles by thermal forces and forces of radiation pressure, analyse the forces acting on the particle in the beam, and uncover the beam structure and intensity to apply for a real-time experiment with XFEL. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only., Published by SPIE, Bellingham, Wash.

Optical trapping of light-absorbing particles in a gaseous environment is governed by a laser-induced photophoretic force, which can be orders of magnitude stronger than the force of radiation-pressure induced by the same light intensity. In spite of many experimental studies, the exact theoretical background underlying the photophoretic force and the prediction of its influence on the particle motion is still in its infancy. Here, we report the results of a quantitative analysis of the photophoretic force and the stiffness of trapping achieved by levitating graphite and carbon-coated glass shells of calibrated sizes in an upright diverging hollow-core vortex beam, which we refer to as an ‘optical funnel’. The measurements of forces were conducted in air at various gas pressures in the range from 5 mbar to 2 bar. The results of these measurements lay the foundation for mapping the optically induced force to the intensity distribution in the trap. The mapping, in turn, provides the necessary information to model flight trajectories of particles of various sizes entering the beam at given initial speed and position relative to the beam axis. Finally, we determined the limits of the parameter space for the particle speed, size, and radial offset to the beam axis, all linked to the laser power and the particular laser beam structure. These results establish the grounds for developing a touch-free optical system for precisely positioning sub-micrometer bioparticles at the focal spot of an x-ray free electron laser, which would significantly enhance the efficiency of studying nanoscale morphology of proteins and biomolecules in femtosecond coherent diffractive imaging experiments.

A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 μm particles can be focused to a full-width at half maximum diameter of 4.2 μm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.

Optical Trapping and Optical Micromanipulation XII, San Diego, California, 9 Aug 2015 - 12 Aug 2015 ; Proceedings of SPIE 9548, 95480H(2015). doi:10.1117/12.2191442, Optical trapping of light-absorbing particles in a gaseous environment is governed by a laser-induced photophoreticforce, which can be orders of magnitude stronger than the force of radiation pressure induced by the same lightintensity. In spite of many experimental studies, the exact theoretical background underlying the photophoreticforce and the prediction of its influence on the particle motion is still in its infancy. Here, we report the results of aquantitative analysis of the photophoretic force and the stiffness of trapping achieved by levitating graphite andcarbon-coated glass shells of calibrated sizes in an upright diverging hollow-core vortex beam, which we refer to asan ‘optical funnel’. The measurements of forces were conducted in air at various gas pressures in the range from 5mbar to 2 bar. The results of these measurements lay the foundation for developing a touch-free optical system forprecisely positioning sub-micrometer bioparticles at the focal spot of an x-ray free electron laser, which wouldsignificantly enhance the efficiency of studying nanoscale morphology of proteins and biomolecules in femtosecondcoherent diffractive imaging experiments., Published by SPIE, Bellingham, Wash.