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For undulator hard X-ray beamlines of a wide energy range, the beam intensity control is often needed to regulate the photon flux impinging on delicate detectors. Despite the capability of state-of-the-art X-ray pixel detectors being greatly advanced recently, the frontier undulator beamlines in synchrotron facilities often carry outstanding photon fluxes that are sometimes too high for certain measurements. Here, we report a developed protocol that allows intensity attenuation of an X-ray beam in the 4-23 keV energy range with flexible attenuation factors; a prototype is installed and tested on the 13A biological small-angle X-ray scattering beamline of the 3 GeV Taiwan Photon Source (TPS) of the National Synchrotron Radiation Research Center, Hsinchu, Taiwan. The intensity attenuation system is modified from a commercially available pneumatically actuated, vacuum-type precision X-ray attenuator of ADC ABS-300; the system provides beam attenuation factors covering 8 orders of intensity attenuation for an X-ray beam in 4-23 keV. This was achieved with selected combinations of 10 sets of metal foils comprising different thicknesses of Al, Ti, Cu, and Ta foils. A user-friendly protocol is established to automatically compare a subscribed attenuation factor with all the possible attenuation factors from the 1024 combinations of the 10 sets of foils in the X-ray energy used, and determine a set of the metal foils having a best-matched attenuation factor. Calculation of beam intensity attenuations with the selected foils is coded with Python and integrated into the Experimental Physics and Industrial Control System (EPICS). The input of an attenuation factor is done through a graphical display of the attenuator system based on the Control System Studio (CSS). The developed X-ray beam attenuation system provides a convenient and intuitive beam intensity control and has the potential to be adopted in future beamlines.

Direct binding of calcium ions (Ca2+) to phospholipid membranes is an unclarified yet critical signaling pathway in diverse Ca2+-regulated cellular phenomena. Here, high-pressure-liquid-chromatography, small-angle X-ray scattering (SAXS), UV–vis absorption, and differential refractive index detections are integrated to probe Ca2+-binding to the zwitterionic lipid membranes in nanodiscs. The responses of the membranes upon Ca2+-binding, in composition and conformation, are quantified through integrated data analysis. The results indicate that Ca2+ binds specifically into the phospholipid headgroup zone, resulting in membrane charging and membrane swelling, with a saturated Ca2+-lipid binding ratio of 1:8. A Ca2+-binding isotherm to the nanodisc is further established and yields an unexpectedly high binding constant K = 4260 M–1 and a leaflet potential of ca. 100 mV based on a modified Gouy–Chapman model. The calcium-lipid binding ratio, however, drops to 40% when the nanodisc undergoes a gel-to-fluid phase...

Membrane thinning that resulted from peptide-binding is observed via temperature dependent small-angle X-ray scattering (SAXS). The result reveals a mean thermal thinning rate of 0.038 Å K-1 for the neat unilamellar vesicles (ULVs) of a zwitterionic phospholipid of 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (diC20:1PC) in the temperature range of 285-312 K. The thinning effect promotes greatly the association between a model antimicrobial peptide melittin and the ULV. Scaling the observed isothermal melittin-ULV bilayer thinning to that measured using low-angle X-ray diffraction from the melittin-multilamellar membranes of defined peptide-to-lipid ratios establishes temperature-dependent binding isotherms χb of the peptide-ULV as a function of free peptide concentration in solution. From the binding isotherms, temperature-dependent peptide-membrane binding constant K(T) is extracted on the basis of a modified Gouy-Chapman model. Changes in K(T) follow the linearized van't Hoff equation ln K(T) ∝ -ΔHT-1 with a constant enthalpy change ΔH = 9.6 kcal mol-1, suggesting an entropy-driven binding process prior to membrane pore formation. Correspondingly, a five-fold enhancement of K is observed in the temperature range studied. The peptide-binding strength is found to follow the growth trend of the membrane thermal thinning rate better than the lipid chain length of the three phosphocholine-based ULVs of diCn:1PC with n = 18, 20, and 22.

Melt-crystallization of syndiotactic polystyrene (sPS) is studied using simultaneous small/wide angle X-ray scattering (SAXS/WAXS). Emergence, intensification, and saturation of WAXS reflections within 120 s at isothermal crystallization temperature Tc = 250 °C after quenching from 300 °C illustrate crystallinity development through successive nucleation of instantaneously stabilized crystallites of lateral (hk0) coherence length Λ ≈ 75 nm. The corresponding SAXS profiles exhibit increased density heterogeneity due to formation of nanograins; fitting of time-resolved SAXS data with a model of arrayed disks reveals slightly thickened (lc = 7.4–8.4 nm) disks of radius R ≈ 9 nm and long period L ≈ 19 nm with polydisperse stacking number slowly enhanced from an average of 1.2 to 2.4. The moderate ratio of Λ/2R ≈ 4 indicates imperfect coalescence of nanograins in the lamellar assembly process. More importantly, with successively increased Tc from 250 to 260 °C the development of density heterogeneity of SAXS invariant Q inv is found to precede that of the WAXS-determined crystallinity Xc increasingly more, suggesting a precursor mesophase with non-crystalline nanograins. Avrami analysis for the developments of Xc and Q inv further reveals the same Avrami exponent n ≈ 3, consistent with heterogeneous nucleation of crystallites; the corresponding Avrami rate constant κ1/n(Tc) extracted from Xc is correlated to the crystal growth rates G(Tc) described by the Hoffman-Lauritzen theory with a common surface-nucleation rate constant Kg. A lateral surface energy σ ≈ 17 mJ m−2 can be deduced on the basis of Kg using previously determined fold surface energy σe ≈ 27 mJ m−2. Correspondingly, κ1/n(Tc) extracted from Qinv is described by a mesophase model proposed by Strobl with a zero-growth temperature of the nanograin mesophase ca. 30 K below the β-crystal melting point of sPS. Together, these results suggest that sPS melt-crystallization proceeds with nucleation of non-crystalline nanograins (SAXS-before-WAXS regime) at the crystal growth front, followed by crystallization of the nanograins with similar kinetics to sustain the growth front. Branching/twisting can occur at the lamellar growth front due to frustrated nanograin alignment, favoring the microscopically observed sheaf-like crystal morphology.

The TPS 13A biological small-angle X-ray scattering (BioSAXS) beamline under construction is equipped with 4-m IU24 undulator for X-rays in the energy range of E = 4-23 keV (covering the K-edge absorptions from Ca to Ru). The beamline aims for cutting-edge researches in biomacromolecular structures and kinetics, in solid and solution, covering a wide length scale from atomic to micrometer scale and time resolution down to μs. To achieve the goals, the beamline integrates double-multilayer and double-crystal monochromators (DMM/DCM) to have an option of high flux of 4 × 1014 photons/s or a high energy resolution of ΔE/E = 1.5×10−4. The DCM together with a horizontal four-bounce crystal collimator (4BCC) will be implanted for low beam divergence to perform 2D ultra-SAXS (USAXS). To keep a common X-ray beam path of the operation modes, DMM and DCM are integrated into one rotating cradle in a same chamber for a common beam exit. Microbeam with reasonable beam divergence for SAXS/WAXS structural mapping will be realized with a virtual source defined by a set of high precision/stability slits and another set of K-B mirrors for a focus at the sample position with a demagnification close to 1.3:1. Two beam position monitors, with intensity and position feedbacks respectively to the virtual source slits and the 4BCC and a vertical deflection mirror, are used mainly to stabilize microbeam intensity and position within 1 μm in the vertical and horizontal directions. This TPS 13A BioSAXS beamline will be commissioning at the end of 2019.

Upon apoptotic stress, Bcl-2 associated X (BAX) protein undergoes conformational changes and oligomerizes, leading to the mitochondrial membrane permeabilization and cell death. While structures of the resultant oligomer have been extensively studied, little is known about the intermediates that describe the reaction pathway from the inactive monomers to activated oligomers. Here we characterize the intermediate structures of BAX using combined small-angle X-ray scattering (SAXS) with on-line gel-filtration and electron spin resonance (ESR). The intermediates, including monomers, dimers, and tetramers, are reconstructed via integrating the SAXS-envelopes and ESR-determined skeleton structures. The hence revealed structures suggest a linear oligomerization of BAX utilizing the extended dimers with the two flexible α6 chains protruded out as ditopic ligands. The results of molecular dynamics simulation also support the ditopic dimer conformation with mobile α6. The ditopic dimers could further wind into a helical rod structure with three dimers in one helical turn. Our results not only reveal the on-pathway intermediates, but also suggest a ditopic oligomerization mechanism that may bridge the observed intermediate structures in solution to the large BAX assemblies lately observed on mitochondria.

Using simultaneously scanning small-angle X-ray scattering (SAXS) and UV–vis absorption with integrated online size exclusion chromatography, supplemental with molecular dynamics simulations, we unveil the long-postulated global structure evolution of a model multidomain protein bovine serum albumin (BSA) during acid-induced unfolding. Our results differentiate three global packing structures of the three molten globule domains of BSA, forming three intermediates I1, I2, and E along the unfolding pathway. The I1–I2 transition, overlooked in all previous studies, involves mainly coordinated reorientations across interconnected molten globule subdomains, and the transition activates a critical pivot domain opening of the protein for entering into the E form, with an unexpectedly large unfolding free energy change of −9.5 kcal mol–1, extracted based on the observed packing structural changes. The revealed local packing flexibility and rigidity of the molten globule domains in the E form elucidate how collect...

To quantitatively correlate membrane thinning with peptide binding affinity, we have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bialyer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer saturates at ∼4 %, significantly lower than the critical thinning of ∼8% (determined via the correspondingly premixed peptide-MLM bilayers) for thermally equilibriated formation of membrane pores, revealing a critical influence of binding affinity for water soluble peptides. Scaling the peptide-ULV bilayer thinning to that of the corresponding peptide-MLM, of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant for melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC may have a same thinning constant upon binding of a same peptide, hence providing a basis in establishing X-ray-based binding isotherms for thermodynamic binding parameters of late stage peptide-membrane interactions prior to pore formation.

With an atomic-force microscope and a grazing-incidence small-angle X-ray scattering we studied ex situ the evolution of hierarchical structures in isothermally annealed ultrathin films of asymmetric polystyrene-block-poly(methyl methacrylate) P(S-b-MMA) that dewetted on polar substrates via a mechanism involving nucleation and growth. Film instability causes the surface to acquire an undulating thickness through incommensurability, producing not only the relief structures on a micrometer scale but also mesophase-separated domains on a nanometer scale. The dewetted morphologies strongly influence the ordering behavior of the nanoscale domains. The noncylindrical nanostructures become stable at the curved edges of the relief microstructures in the destabilized P(S-b-MMA) films, for which a preferential wetting of the PS block with the free surface is prohibited. Additionally, the shape of relief structures as result of film instability correlates with the formation of mesophase-separated nanodomains. At early stages of film instability, the formation of parallel-oriented PMMA cylindrical nanodomains increases the deformation energy and it further persists to force the shape of relief structures between irregular holes to have a facet-wedge shape. However, those relief structures are expected to be not at equilibrium. At high temperatures, the relief structures between irregular holes progressively developed to form hemispherical-cap drops accompanied by a transformation of cylindrical into noncylindrical nanodomains at curved surfaces.

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 realizedviaa 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 illustratedviaseveral 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 underin situdrawing, (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 formedin situat the air–water interface.

At the National Synchrotron Radiation Research Center (NSRRC), which operates a 1.5 GeV storage ring, a dedicated small-angle X-ray scattering (SAXS) beamline has been installed with an in-achromat superconducting wiggler insertion device of peak magnetic field 3.1 T. The vertical beam divergence from the X-ray source is reduced significantly by a collimating mirror. Subsequently the beam is selectively monochromated by a double Si(111) crystal monochromator with high energy resolution (DeltaE/E approximately 2 x 10(-4)) in the energy range 5-23 keV, or by a double Mo/B4C multilayer monochromator for 10-30 times higher flux ( approximately 10(11) photons s(-1)) in the 6-15 keV range. These two monochromators are incorporated into one rotating cradle for fast exchange. The monochromated beam is focused by a toroidal mirror with 1:1 focusing for a small beam divergence and a beam size of approximately 0.9 mm x 0.3 mm (horizontal x vertical) at the focus point located 26.5 m from the radiation source. A plane mirror installed after the toroidal mirror is selectively used to deflect the beam downwards for grazing-incidence SAXS (GISAXS) from liquid surfaces. Two online beam-position monitors separated by 8 m provide an efficient feedback control for an overall beam-position stability in the 10 microm range. The beam features measured, including the flux density, energy resolution, size and divergence, are consistent with those calculated using the ray-tracing program SHADOW. With the deflectable beam of relatively high energy resolution and high flux, the new beamline meets the requirements for a wide range of SAXS applications, including anomalous SAXS for multiphase nanoparticles (e.g. semiconductor core-shell quantum dots) and GISAXS from liquid surfaces.

Melting of the semicrystalline structure of native tapioca starch granules is correlated to solution viscosity for elucidating gelatinization and gelation processes., Melting of native tapioca starch granules in aqueous pastes upon heating is observed in situ using simultaneous small- and wide-angle X-ray scattering (SAXS/WAXS) and solution viscometry. Correlated structure and viscosity changes suggest closely associated amylose and amylopectin chains in the semicrystalline layers, and the release of amylose chains for enhanced solution viscosity occurs largely after melting of the semicrystalline structure. Before melting, WAXS results reveal mixed crystals of A- and B-types (∼4:1 by weight), whereas SAXS results indicate that the semicrystalline layers are composed of lamellar blocklets of ca 43 nm domain size, with polydisperse crystalline (≃7.5 nm) and amorphous (≃1.1 nm) layers alternatively assembled into a lamellar spacing of ≃8.6 nm with 20% polydispersity. Upon melting, the semicrystalline lamellae disintegrate into disperse and molten amylopectin nanoclusters with dissolved and partially untangled amylose chains in the aqueous matrix which leads to increased solution viscosity. During subsequent cooling, gelation starts at around 347 K; successively increased solution viscosity coincides with the development of nanocluster aggregation to a fractal dimension ≃2.3 at 303 K, signifying increasing intercluster association through collapsed amylose chains owing to decreased solvency of the aqueous medium with decreasing temperature.