Ying-Huang Lai

A small/wide-angle X-ray scattering instrument for structural characterization of air-liquid interfaces, thin films and bulk specimens

At the National Synchrotron Radiation Research Center, a small/wide-angle X-ray scattering (SAXS/WAXS) instrument has been installed at the BL23A beamline with a superconducting wiggler insertion device. This beamline is equipped with double Si(111) crystal and double Mo/B4C multilayer monochromators, and an Si-based plane mirror that can selectively deflect the beam downwards for grazing-incidence SAXS (GISAXS) studies of air–liquid or liquid–liquid interfaces. The SAXS/WAXS instrument, situated in an experimental hutch, comprises collimation, sample and post-sample stages. Pinholes and slits have been incorporated into the beam collimation system spanning a distance of ∼5 m. The sample stage can accommodate various sample geometries for air–liquid interfaces, thin films, and solution and solid samples. The post-sample section consists of a 1 m WAXS section with two linear gas detectors, a vacuum bellows (1–4 m), a two-beamstop system and the SAXS detector system, all situated on a motorized optical bench for motion in six degrees of freedom. In particular, the vacuum bellows of a large inner diameter (260 mm) provides continuous changes of the sample-to-detector distance under vacuum. Synchronized SAXS and WAXS measurements are realized via a data-acquisition protocol that can integrate the two linear gas detectors for WAXS and the area detector for SAXS (gas type or Mar165 CCD); the protocol also incorporates sample changing and temperature control for programmable data collection. The performance of the instrument is illustrated via several different measurements, including (1) simultaneous SAXS/WAXS and differential scanning calorimetry for polymer crystallization, (2) structural evolution with a large ordering spacing of ∼250 nm in a supramolecular complex, (3) SAXS for polymer blends under in situ drawing, (4) SAXS and anomalous SAXS for unilamellar lipid vesicles and metalloprotein solutions, (5) anomalous GISAXS for oriented membranes of Br-labeled lipids embedded with peptides, and (6) GISAXS for silicate films formed in situ at the air–water interface.

X-ray beamlines for structural studies at the NSRRC superconducting wavelength shifter

Using a superconducting-wavelength-shifter X-ray source with a photon flux density of 10(11)-10(13) photons s(-1) mrad(-1) (0.1% bandwidth)(-1) (200 mA)(-1) in the energy range 5-35 keV, three hard X-ray beamlines, BL01A, BL01B and BL01C, have been designed and constructed at the 1.5 GeV storage ring of the National Synchrotron Radiation Research Center (NSRRC). These have been designed for structure-related research using X-ray imaging, absorption, scattering and diffraction. The branch beamline BL01A, which has an unmonochromatized beam, is suitable for phase-contrast X-ray imaging with a spatial resolution of 1 microm and an imaging efficiency of one frame per 10 ms. The main beamline BL01B has 1:1 beam focusing and a medium energy resolution of approximately 10(-3). It has been designed for small-angle X-ray scattering and transmission X-ray microscopy, used, respectively, in anomalous scattering and nanophase-contrast imaging with 30 nm spatial resolution. Finally, the branch beamline BL01C, which features collimating and focusing mirrors and a double-crystal monochromator for a high energy resolution of approximately 10(-4), has been designed for X-ray absorption spectroscopy and high-resolution powder X-ray diffraction. These instruments, providing complementary tools for studying multiphase structures, have opened up a new research trend of integrated structural study at the NSRRC, especially in biology and materials. Examples illustrating the performances of the beamlines and the instruments installed are presented.

An instrument for time-resolved and anomalous simultaneous small- and wide-angle X-ray scattering (SWAXS) at NSRRC

A SWAXS (small- and wide-angle X-ray scattering) instrument was recently installed at the wiggler beamline BL17B3 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The instrument, which is designed for studies of static and dynamic nanostructures and correlations between the nano (or meso) structure (SAXS) and crystalline structure (WAXS), provides a flux of 1010–1011 photon s−1 at the sample at energies between 5 and 14 keV. With a SAXS area detector and a WAXS linear detector connected to two data acquisition systems operated in master–slave mode, the instrument allows one to perform time-resolved as well as anomalous scattering measurements. Data reduction algorithms have been developed for rapid processing of the large SWAXS data sets collected during time-resolved measurements. The performance of the instrument is illustrated by examples taken from different classes of ongoing projects: (i) time-resolved SAXS/WAXS/differential scanning calorimetry (DSC) with a time resolution of 10 s on a semicrystalline poly(hexamethylene terephthalate) sample, (ii) anomalous SAXS/WAXS measurements on a nanoparticulate PtRu catalyst, and (iii) grazing-incidence SAXS of a monolayer of oriented semiconductor quantum wires, and humidity-controlled ordering of Alamethicin peptides embedded in an oriented lipid membrane.

Anomalous small- and wide-angle X-ray scattering and X-ray absorption spectroscopy for Pt and Pt-Ru nanoparticles

We have characterized the structures of two kinds of catalytic nanoparticles of Pt and Pt–Ru, using anomalous small-angle X-ray scattering (ASAXS), anomalous wide-angle X-ray scattering (AWAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy. With several X-ray energies near the Pt LIII edge, the AWAXS data reveal a face-centered cubic (f.c.c.) crystalline structure for Pt nanoparticles supported on carbon black, whereas the ASAXS data characterize the monometallic nanoparticles as polydisperse spheres with a mean size of 23 A and a size distribution of 20%. With similar X-ray energies, ASAXS and AWAXS data for the Pt–Ru nanoparticles indicate that they have a mean size of ~29 A and a slightly Pt-rich core that can be characterized by an f.c.c. crystalline structure similar to that of the pure Pt nanoparticles. The coordination numbers of the bimetallic nanoparticles extracted from the EXAFS data, collected at the Pt LIII edge and Ru K edge, also reveal a consistent structure of largely, but not completely, intermixed Pt and Ru atoms in the nanoparticles.

Global and Local Structural Changes of Cytochrome c and Lysozyme Characterized by a Multigroup Unfolding Process

Equilibrium unfolding behaviors of cytochrome c and lysozyme induced by the presence of urea (0-10 M) as well as changes in temperature (295-363 K) or pH (1.8-7) are examined via small-angle x-ray scattering and spectroscopic techniques, including circular dichroism and optical absorption. Denaturant and temperature effects are incorporated into the free energy expression for a general multigroup unfolding process. Results indicate that there are at least four unfolding groups in the temperature-, urea-, or pH-induced unfolding of cytochrome c: two of these are related to the prosthetic heme group, and the other two correspond, respectively, to the unfolding of alpha-helices and global changes in protein morphology that are largely unaccounted for by the first two groups. In contrast, the unfolding of lysozyme approximately follows a simple one-group process. A modified mean-field Ising model is adopted for a coherent description of the unfolding behaviors observed. Thermodynamic parameters extracted from simple denaturing processes, on the basis of the Ising model, can closely predict unfolding behaviors of the proteins in compounded denaturing environments.

Resonant X-Ray Scattering and Absorption for the Global and Local Structures of Cu-modified Metallothioneins in Solution

With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing Cu(I) or Cu(II) ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in Cu(I)-MT and Cu(II)-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of Cu(II)-MT resembles that of the native MT, whereas Cu(I)-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for Cu(II)-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both Cu(I) and Cu(II) ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques.

Charge interaction and temperature effects on the solution structure of lysozyme as revealed by small-angle X-ray scattering

We have studied the structure of lysozyme as influenced by solution environment using small-angle X-ray scattering (SAXS). With an ellipsoid form factor and a structure factor derived using the mean spherical approximation to account for the electrostatic repulsion of lysozyme, we have extracted detailed structural information about the protein in aqueous solutions, including the size, shape, and net charge number. The SAXS data analysis shows that lysozyme in pure water, expressing an averaged net charge number of ~6, folds to an ellipsoid-like shape with a radius of gyration Rg = 16.6 A. Temperature-dependent SAXS for lysozyme in a buffer solution in which charge repulsion has been eliminated suggests that the protein may thermally unfold gradually along a preferred direction from the ellipsoidal shape with an aspect ratio of p ≃ 2 at 303 K to an elongated shape with p ≃ 3 at 343 K. The structural parameters of the unfolded lysozyme obtained using model fitting are compared with the envelope morphology simulated using a dummy-residues model. From the evolution of the volume of lysozyme during the thermal unfolding process, we deduce a free-energy profile for the protein thermally unfolded in water using a modified Ising model on the basis of a mean field approximation.

Anomalous SAXS for the morphology and metal content of a metallothionein in a metal-atom replacement

1 all play. Although we are all using the same ball, confusion reigns because we each play the game according to our own set of rules. A good chemical model requires well defined concepts that lead to quantitative predictions. The ionic model meets these criteria. The electroneutrality rule (the sum of all atomic valences in a compound is zero) is the only assumption the model makes and it applies to all compounds that obey this rule (mostly inorganic compounds, but also aqueous chemistry, hence much of biological chemistry). From the Coulomb field of the ionic model one can derive rigorous concepts of atom, bond, atomic valence, bond valence and electronegativity as well as Lewis acid and base strength. The result is a predictive model expressed in terms of the familiar chemical concept of localized bonds linking nearest-neighbour atoms.