Hydroxyethyl cellulose matrix applied to serial crystallography

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3,000 diffraction images. ? 2017 The Author(s).113Ysciescopu

Atomic resolution structure of serine protease proteinase K at ambient temperature

Atomic resolution structures (beyond 1.20 ?) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 ? resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites. ? 2017 The Author(s).114Ysciescopu

Soft X-ray harmonic comb from relativistic electron spikes

We demonstrate a new high-order harmonic generation mechanism reaching the `water window' spectral region in experiments with multi-terawatt femtosecond lasers irradiating gas jets. A few hundred harmonic orders are resolved, giving uJ/sr pulses. Harmonics are collectively emitted by an oscillating electron spike formed at the joint of the boundaries of a cavity and bow wave created by a relativistically self-focusing laser in underdense plasma. The spike sharpness and stability are explained by catastrophe theory. The mechanism is corroborated by particle-in-cell simulations

Coulomb implosion mechanism of negative ion acceleration in laser plasmas

Coulomb implosion mechanism of the negatively charged ion acceleration in laser plasmas is proposed. When a cluster target is irradiated by an intense laser pulse and the Coulomb explosion of positively charged ions occurs, the negative ions are accelerated inward. The maximum energy of negative ions is several times lower than that of positive ions. The theoretical description and Particle-in-Cell simulation of the Coulomb implosion mechanism and the evidence of the negative ion acceleration in the experiments on the high intensity laser pulse interaction with the cluster targets are presented.Comment: 4 page

X-ray harmonic comb from relativistic electron spikes

X-ray devices are far superior to optical ones for providing nanometre spatial and attosecond temporal resolutions. Such resolution is indispensable in biology, medicine, physics, material sciences, and their applications. A bright ultrafast coherent X-ray source is highly desirable, for example, for the diffractive imaging of individual large molecules, viruses, or cells. Here we demonstrate experimentally a new compact X-ray source involving high-order harmonics produced by a relativistic-irradiance femtosecond laser in a gas target. In our first implementation using a 9 Terawatt laser, coherent soft X-rays are emitted with a comb-like spectrum reaching the 'water window' range. The generation mechanism is robust being based on phenomena inherent in relativistic laser plasmas: self-focusing, nonlinear wave generation accompanied by electron density singularities, and collective radiation by a compact electric charge. The formation of singularities (electron density spikes) is described by the elegant mathematical catastrophe theory, which explains sudden changes in various complex systems, from physics to social sciences. The new X-ray source has advantageous scalings, as the maximum harmonic order is proportional to the cube of the laser amplitude enhanced by relativistic self-focusing in plasma. This allows straightforward extension of the coherent X-ray generation to the keV and tens of keV spectral regions. The implemented X-ray source is remarkably easily accessible: the requirements for the laser can be met in a university-scale laboratory, the gas jet is a replenishable debris-free target, and the harmonics emanate directly from the gas jet without additional devices. Our results open the way to a compact coherent ultrashort brilliant X-ray source with single shot and high-repetition rate capabilities, suitable for numerous applications and diagnostics in many research fields

Single-shot 3D coherent diffractive imaging of core-shell nanoparticles with elemental specificity

We report 3D coherent diffractive imaging (CDI) of Au/Pd core-shell nanoparticles with 6.1 nm spatial resolution with elemental specificity. We measured single-shot diffraction patterns of the nanoparticles using intense x-ray free electron laser pulses. By exploiting the curvature of the Ewald sphere and the symmetry of the nanoparticle, we reconstructed the 3D electron density of 34 core-shell structures from these diffraction patterns. To extract 3D structural information beyond the diffraction signal, we implemented a super-resolution technique by taking advantage of CDI's quantitative reconstruction capabilities. We used high-resolution model fitting to determine the Au core size and the Pd shell thickness to be 65.0 +/- 1.0 nm and 4.0 +/- 0.5 nm, respectively. We also identified the 3D elemental distribution inside the nanoparticles with an accuracy of 3%. To further examine the model fitting procedure, we simulated noisy diffraction patterns from a Au/Pd core-shell model and a solid Au model and confirmed the validity of the method. We anticipate this super-resolution CDI method can be generally used for quantitative 3D imaging of symmetrical nanostructures with elemental specificity.111Ysciescopu

Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length

Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression

High order harmonics from relativistic electron spikes

A new regime of relativistic high-order harmonic generation is discovered [Phys. Rev. Lett. 108, 135004 (2012)]. Multi-terawatt relativistic-irradiance (>1018 W/cm2) femtosecond (~30-50 fs) lasers focused to underdense (few×1019 cm-3) plasma formed in gas jet targets produce comb-like spectra with hundreds of even and odd harmonic orders reaching the photon energy of 360 eV, including the 'water window' spectral range. Harmonics are generated by either linearly or circularly polarized pulses from the J-KAREN (KPSI, JAEA) and Astra Gemini (CLF, RAL, UK) lasers. The photon number scalability has been demonstrated with a 120 TW laser producing 40 μJ/sr per harmonic at 120 eV. The experimental results are explained using particle-in-cell (PIC) simulations and catastrophe theory. A new mechanism of harmonic generation by sharp, structurally stable, oscillating electron spikes at the joint of boundaries of wake and bow waves excited by a laser pulse is introduced. In this paper detailed descriptions of the experiments, simulations and model are provided and new features are shown, including data obtained with a two-channel spectrograph, harmonic generation by circularly polarized laser pulses and angular distribution

Oil-free hyaluronic acid matrix for serial femtosecond crystallography

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-angstrom resolution.open111011sciescopu

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 angstrom is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.open112633sciescopu