Showing papers by "Yasumasa Joti published in 2018"

A soft X‐ray free‐electron laser beamline at SACLA: the light source, photon beamline and experimental station

Abstract: The design and performance of a soft X-ray free-electron laser (FEL) beamline of the SPring-8 Compact free-electron LAser (SACLA) are described. The SPring-8 Compact SASE Source test accelerator, a prototype machine of SACLA, was relocated to the SACLA undulator hall for dedicated use for the soft X-ray FEL beamline. Since the accelerator is operated independently of the SACLA main linac that drives the two hard X-ray beamlines, it is possible to produce both soft and hard X-ray FEL simultaneously. The FEL pulse energy reached 110 µJ at a wavelength of 12.4 nm (i.e. photon energy of 100 eV) with an electron beam energy of 780 MeV.

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

Abstract: 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.

Software for the data analysis of the arrival-timing monitor at SACLA.

Abstract: X-ray free-electron laser (XFEL) pulses from SPring-8 Angstrom Compact free-electron LAser (SACLA) with a temporal duration of <10 fs have provided a variety of benefits in scientific research. In a previous study, an arrival-timing monitor was developed to improve the temporal resolution in pump-probe experiments at beamline 3 by rearranging data in the order of the arrival-timing jitter between the XFEL and the synchronized optical laser pulses. This paper presents Timing Monitor Analyzer (TMA), a software package by which users can conveniently obtain arrival-timing data in the analysis environment at SACLA. The package is composed of offline tools that pull stored data from cache storage, and online tools that pull data from a data-handling server in semi-real time during beam time. Users can select the most suitable tool for their purpose, and share the results through a network connection between the offline and online analysis environments.

Gaussian mixture model for coarse-grained modeling from XFEL

Abstract: We explore the advantage of Gaussian mixture model (GMM) for interpretation of single particle diffraction patterns from X-ray free electron laser (XFEL) experiments. GMM approximates a biomolecular shape by the superposition of Gaussian distributions. As the Fourier transformation of GMM can be quickly performed, we can efficiently simulate XFEL diffraction patterns from approximated structure models. We report that the resolution that GMM can accurately reproduce is proportional to the cubic root of the number of Gaussians used in the modeling. This behavior can be attributed to the correspondence between the number of adjustable parameters in GMM and the amount of sampling points in diffraction space. Furthermore, GMMs can successfully be used to perform angular assignment and to detect conformational variation. These results demonstrate that GMMs serve as useful coarse-grained models for hybrid approach in XFEL single particle experiments.

Strategy for Stabilization of CutA1 Proteins Due to Ion–Ion Interactions at Temperatures of over 100 °C

Abstract: In order to elucidate the contribution of charged residues to protein stabilization at temperatures of over 100 °C, we constructed many mutants of the CutA1 protein ( EcCutA1) from Escherichia coli. The goal was to see if one can achieve the same stability as for a CutA1 from hyperthermophile Pyrococcus horikoshii that has the denaturation temperature near 150 °C. The hydrophobic mutant of EcCutA1 ( Ec0VV) with denaturation temperature ( Td) of 113.2 °C was used as a template for mutations. The highest Td of Ec0VV mutants substituted by a single charged residue was 118.4 °C. Multiple ion mutants were also constructed by combination of single mutants and found to have an increased thermostability. The highest stability of multiple mutants was a mutant substituted by nine charged residues that had a Td of 142.2 °C. To evaluate the energy of ion-ion interactions of mutant proteins, we used the structural ensemble obtained by a molecular dynamics simulation at 300 K. The Td of ionic mutants linearly increases with the increments of the computed energy of ion-ion interactions for ionic mutant proteins even up to the temperatures near 140 °C, suggesting that ion-ion interactions cumulatively contribute to the stabilization of a protein at high temperatures.

Ion-ion interactions in the denatured state contribute to the stabilization of CutA1 proteins.

Abstract: In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (Td) = 85.6 °C, a hydrophobic mutant (Ec0VV) with Td = 113.3 °C, and an ionic mutant (Ec0VV_6) with Td = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

Development of Multilayer Focusing Mirror System for XFEL CDI Experiments of Biological Particles

Abstract: A single shot coherent diffractive imaging (CDI) using an X-ray free-electron laser (XFEL) enable us to visualize structures of a specimen without radiation damage owing to the femtosecond pulse nature. Living cells have been observed by pulsed coherent X-ray solution scattering (PCXSS) [1] using XFEL pulses from the SPring-8 Angstrom Compact free-electron LAser (SACLA). Although the resolution of a few tens nanometer has been achieved, there is a growing demand of resolution as high as single nanometer range. In order to meet the demand, we developed high-resolution CDI system employed multilayer focusing mirrors which has a high throughput and short focal lengths to further increase fluence at the sample plane.

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in structured biomacromolecules

Abstract: The clock of life ticks as fast as how efficiently proteins could perform their functional dynamics. Protein complexes execute functions via several large-scale intrinsic motions across multiple conformational states, which occur at a timescale of nano- to milliseconds for well-folded proteins. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to time and size these motions, though barely to their slowest ends. Here, we convert a simple elastic network model (ENM), which takes a few seconds (ubiquitin) to hours (ribosome) for the analysis, into a molecular timer and sizer to gauge the slowest functional motions of proteins. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener-Khintchine theorem (WKT) are used to define the "time-periods", t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective "time-periods" are mapped to the eigenvalues (λENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 86.9λENM-1.9 and σ2(A2) = 46.1λENM-2.5 are established allowing the characterization of the time scales of Nuclear Magnetic Resonance (NMR)-solved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in the ribosome

Abstract: The clock of life ticks as fast as how efficiently proteins could perform their functional dynamics. Protein complexes execute functions via several large-scale intrinsic motions across multiple conformational states, which occur at a timescale of nano- to milliseconds for well-folded proteins. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to time and size these motions, though barely to their slowest ends. Here, we convert a simple elastic network model (ENM), which takes a few seconds (ubiquitin) to hours (ribosome) for the analysis, into a molecular timer and sizer to gauge the slowest functional motions of proteins. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener-Khintchine theorem (WKT) are used to define the "time-periods", t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective "time-periods" are mapped to the eigenvalues (λ ENM ) of the corresponding ENM modes. Thus, the power laws t (ns) = 86.9λ ENM -1.9 and σ 2 (A 2 ) = 46.1λ ENM -2.5 are established allowing the characterization of the time scales of Nuclear Magnetic Resonance (NMR)-solved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.