The ultrafast evolution of microstructure is key to understanding high-pressure and strain-rate phenomena. However, the visualization of lattice dynamics at scales commensurate with those of ...atomistic simulations has been challenging. Here, we report femtosecond x-ray diffraction measurements unveiling the response of copper to laser shock-compression at peak normal elastic stresses of ~73 gigapascals (GPa) and strain rates of 10⁹ per second. We capture the evolution of the lattice from a one-dimensional (1D) elastic to a 3D plastically relaxed state within a few tens of picoseconds, after reaching shear stresses of 18 GPa. Our in situ high-precision measurement of material strength at spatial (< 1 micrometer) and temporal (< 50 picoseconds) scales provides a direct comparison with multimillion-atom molecular dynamics simulations.
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein ...microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.
Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four ...cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.
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•MBP-1 toxicity is restrained via crystallization in eosinophil secretory granules•The nanocrystals are amenable to structural characterization using XFEL radiation•MBP-1 amyloidogenic aggregation mediates toxicity toward pathogens and host cells•Bulky extracellular plaques limit immunopathology in eosinophil-infiltrated organs
MBP-1 is a powerful toxin secreted by eosinophils as part of the innate immune response against pathogens that can also cause tissue damage in eosinophilic diseases. Soragni et al. show how MBP-1 crystallization and amyloidogenic aggregation regulate its toxicity toward pathogens and host cells.
Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP ...with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal ...disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
The Coherent X‐ray Imaging (CXI) instrument specializes in hard X‐ray, in‐vacuum, high power density experiments in all areas of science. Two main sample chambers, one containing a 100 nm focus and ...one a 1 µm focus, are available, each with multiple diagnostics, sample injection, pump–probe and detector capabilities. The flexibility of CXI has enabled it to host a diverse range of experiments, from biological to extreme matter.
X-ray scattering is typically used as a weak linear atomic-scale probe of matter. At high intensities, such as produced at free-electron lasers, nonlinearities can become important, and the probe may ...no longer be considered weak. Here we report the observation of one of the most fundamental nonlinear X-ray-matter interactions: the concerted nonlinear Compton scattering of two identical hard X-ray photons producing a single higher-energy photon. The X-ray intensity reached 4 × 1020 W cm-2, corresponding to an electric field well above the atomic unit of strength and within almost four orders of magnitude of the quantum-electrodynamic critical field. We measure a signal from solid beryllium that scales quadratically in intensity, consistent with simultaneous non-resonant two-photon scattering from nearly-free electrons. The high-energy photons show an anomalously large redshift that is incompatible with a free-electron approximation for the ground-state electron distribution, suggesting an enhanced nonlinearity for scattering at large momentum transfer.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary ...increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
We present a proof-of-concept three-dimensional reconstruction of the giant mimivirus particle from experimentally measured diffraction patterns from an x-ray free-electron laser. Three-dimensional ...imaging requires the assembly of many two-dimensional patterns into an internally consistent Fourier volume. Since each particle is randomly oriented when exposed to the x-ray pulse, relative orientations have to be retrieved from the diffraction data alone. We achieve this with a modified version of the expand, maximize and compress algorithm and validate our result using new methods.
In nanotechnology, strategies for the creation and manipulation of nanoparticles in the gas phase are critically important for surface modification and substrate-free characterization. Recent ...coherent diffractive imaging with intense femtosecond X-ray pulses has verified the capability of single-shot imaging of nanoscale objects at suboptical resolutions beyond the radiation-induced damage threshold. By intercepting electrospray-generated particles with a single 15 femtosecond soft-X-ray pulse, we demonstrate diffractive imaging of a nanoscale specimen in free flight for the first time, an important step toward imaging uncrystallized biomolecules.
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