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Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.

KAMO and BLEND provide particularly effective tools to automatically manage the merging of large numbers of data sets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending BLEND to support additional clustering options such as the use of more accurate cell distance metrics and the use of reflection-intensity correlation coefficients to infer `distances' among sets of reflections. This increases the sensitivity to differences in unit-cell parameters and allows clustering to assemble nearly complete data sets on the basis of intensity or amplitude differences. If the data sets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. When starting from incomplete data sets, one applies KAMO twice, first using unit-cell parameters. In this step, either the simple cell vector distance of the original BLEND or the more sensitive NCDist is used. This step tends to find clusters of sufficient size such that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between reflections with a common hkl to merge clusters in a way that is sensitive to structural differences that may not have perturbed the unit-cell parameters sufficiently to make meaningful clusters. Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into categories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent of one another, it should be possible to greatly improve the efficacy of data-clustering software by using a multi-stage partitioning strategy. Here, one possible approach to multi-stage data clustering is demonstrated. The strategy is to use unit-cell clustering until the merged data are sufficiently complete and then to use intensity-based clustering. Using this strategy, it is demonstrated that it is possible to accurately cluster data sets from crystals that have subtle differences.

The Earth has undergone at least four great glaciations, during which the ice layers and the glaciers have expanded all over the planet, corresponding to significant drops in global temperatures th...

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5–30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s−1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s−1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods – serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362–1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238–246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

KAMO and Blend provide particularly effective tools to manage automatically the merging of large numbers of datasets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending Blend to support additional clustering options such as use of more accurate cell distance metrics and use of reflection-intensity correlation coefficients to infer “distances” among sets of reflec- tions. This increases the sensitivity to differences in unit cell parameters and allows for clustering to assemble nearly complete datasets on the basis of intensity or ampli- tude differences. If datasets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. If starting from incomplete datasets, one applies KAMO twice, first using cell parameters. In this step we use either the simple cell vector distance of the original Blend, or we use the more sensi- tive NCDist. This step tends to find clusters of sufficient size so that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between the reflections having a common hkl to merge clusters in a way sensitive to structural differences that may not have perturbed the cell parameters sufficiently to make meaningful clusters.Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into cate- gories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent from one another, it should be possible to greatly improve the efficacy of data clustering software by using a multi-stage partitioning strategy. Here, we have demonstrated one possible approach to multi-stage data clustering. Our strategy is to use unit-cell clustering until merged data is sufficiently complete then to use intensity-based clustering. We have demonstrated that, using this strategy, we are able to accurately cluster datasets from crystals that have subtle differences.

Macromolecular Crystallography (MX) is becoming a big data science straining the capabilities of computers and networks. New techniques of serial crystallography are allowing new science to be done but they are increasing the heterogeneity of the data that must be handled. Two new beamlines at the National Synchrotron Light Source-II, for Frontier Macromolecular Crystallography (FMX) and for highly Automated Macromolecular Crystallography (AMX), are beginning user operation in 2016, and are dealing with these big data issues. This work is contributing to a worldwide High Data-Rate Macromolecular Crystallography (HDRMX) collaboration among crystallographic software developers, beamline scientists and controls people to resolve these issues as new high-brightness beamlines and new fast X-ray detectors come into increasing use.

We present the final design of the x-ray optics and experimental stations of two macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source-II. The microfocusing FMX beamline will deliver a flux of ∼5×1012 ph/s at 1 A into a 1 – 20 µm spot, its flux density surpassing current MX beamlines by up to two orders of magnitude. It covers an energy range from 5 – 30 keV. The highly automated AMX beamline is optimized for high throughput, with beam sizes from 4 – 100 µm, an energy range of 5 – 18 keV and a flux at 1 A of ∼1013 ph/s. A focus in designing the beamlines lay on achieving high beam stability, for example by implementing a horizontal bounce double crystal monochromator at FMX. A combination of compound refractive lenses and bimorph mirror optics at FMX supports rapid beam size changes. Central components of the in-house developed experimental stations are horizontal axis goniometers with a target sphere of confusion of 100 nm, piezo-slits for dynamic beam size changes during...

The instrumentation and methods available for collecting almost simultaneous single-crystal electronic absorption correlated with X-ray diffraction data at NSLS beamline X26-C are reviewed, as well as a very brief outline of its Raman spectroscopy capability., The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.

The collection of absorption and Raman spectroscopic data correlated with X-ray diffraction data allows investigators to understand the atomic structure as well as the electronic and vibrational characteristics of their samples, to identify transiently formed intermediates and to explore mechanistic questions. Raman spectroscopy instrumentation at beamline X26-C at the NSLS is currently available to the general user population., Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data.

The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial [1]. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

This manuscript chronicles the evolution of software used originally to control a diffractometer at a macromolecular crystallography beamline. The system has been augmented and rewritten. A modular and carefully organized suite of programs now handles the whole experimental environment from a single vantage point. It provides automatic logging of the experiment and communication with the user, all the way from an initial proposal to perform the work to the end of data collection. This has included construction of a relational database to organize all details of the experiment and incorporation of a robotic specimen changer to provide automation for high-throughput applications.

Beam line X29 of the National Synchrotron Light Source (NSLS) has been operating for a year for macromolecular crystallography (PX) research, employing a mini-gap, in-vacuum undulator as its X-ray source. Development of this facility involved a close collaboration among the groups of Mark Chance (Case Proteomics Center, Case Western Reserve Univ. [CWR]), Erik Johnson [NSLS], and Robert Sweet (Macromolecular Crystallography Research Resource [PXRR] of BNL's Biology Dept.). There were several joint objectives and complexities to this project. Firstly, we hoped to provide an undulator-based beam line with the attractive features of high collimation and high flux for macromolecular crystallography at the NSLS.

Triplet phases recorded from insulin crystals were used to measure the improvement of phases during model refinement and to quantify the contribution made by each step in the refinement. Conventional amplitude data were recorded to 1.5 A resolution from rhombohedral pig insulin crystals using 1.54 A Cu Kalpha radiation. An initial atomic model and starting phases were obtained from a published structure and the atomic model was refined against the amplitude data using CNS. The refined phases were compared with 800 triplet phases that were measured from similar crystals using a three-beam interference technique and 1.1 A wavelength synchrotron radiation. The solvent region was improved further using a novel density-modification procedure. Calculated triplet phases were obtained from the model after each step in the refinement and were compared with the recorded triplet phases. The average difference between the recorded triplet phases and the calculated triplet phases was used as an unbiased measure of the correctness of the model at each stage in the refinement. The average individual phase error was estimated from discrepancies from triplet phases after each refinement step. Conventional atomic refinement of an approximate starting model reduced the average individual phase error from 21.6 to 14.7 degrees. Improvement of the solvent region, including the difference-map flattening procedure, reduced the individual phase error by a further 2.6 degrees. Modeling the discrete disorder of four amino acids accounted for an additional 0.5 degrees improvement and the final individual phase error was 11.6 degrees.

Macromolecular crystallographic (MX) structure determination at synchrotron radiation sources has the potential to advance significantly through use of X-ray beams of one micron or smaller cross-sections. Recently, the MX Frontiers at the One Micron Scale Workshop explored structural biology scientific opportunities made possible through the use of micro-beams, and anticipated technical challenges for developers of MX beamlines at the National Synchrotron Light Source II (NSLS-II). More than 100 attendees participated in the workshop, which included one-and-a-half days of lectures, discussions, and a semi-formal poster session on July 23–24, 2009, at Brookhaven National Laboratory (BNL).

The effects of regulatory amounts of Ca 2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied ±Ca 2+ in 41.6% 2 H 2 O buffers in which protonated subunits were rendered “invisible”. We found that the radius of gyration ( R g ) of TnI decreased by ≈10% upon addition of regulatory Ca 2+ indicating that it was significantly more compact in the presence of Ca 2+ . The apparent cross-sectional radius of gyration ( R c ) of TnI increased by about 9% when regulatory Ca 2+ was bound to TnC. Modeling studies showed that the high- Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca 2+ addition decreased the center of mass separation by about 1.5 nm. The R g of TnI, its high- Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca 2+ ) TnC·TnI complex. The R g of TnC indicated that it was elongate in situ . The R g of TnC was not sensitive to the Ca 2+ occupancy of its regulatory sites. However, R c increased upon Ca 2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca 2+ -induced changes in TnC.

The coronal structure of starlike block ionomer micelles has been probed directly by small-angle neutron scattering (SANS), using specific contrast labeling of different regions of the corona. Solutions of PS-b-dPS-b-PS-b-PACs starlike block ionomer micelles in toluene are studied; contrast is provided by perdeuterated polystyrene labels situated at different distances from the ionic core. For deuterated labels situated at 390 PS units from the core, the scattered intensity is found to scale as I ∼ q-1.7 at intermediate q, suggesting a semidilute environment for the labels and local swollen coil behavior. For labels 180 PS units from the core, steric perturbations are more extensive than those found under semidilute conditions, such that single chain scaling is not observed at any length scale; scattered intensity is found to scale as I ∼ q-1.3 at intermediate q. The stiffness of coronal chains next to the core is even greater, as indicated by I ∼ q-1.0 scaling for dPS labels attached directly to the ioni...

We have investigated the coarsening process of domains having a cubic lattice in a nonionic surfactant system using a time-resolved small-angle neutron scattering technique. The obtained time evolution of scattering patterns showed growing large domains with strong diffraction intensity fluctuation and shrinking small domains obeying dynamical scaling law. This bimodal domain coarsening suggests an anomalous domain coarsening. During the coarsening the cubic lattice was distorted about 10%, which is probably due to the adjustment of the adjacent lattice at boundary. This elasticity effect probably brings about the observed anomalous domain coarsening.

We have investigated the stability of a cubic lattice having a periodic-minimal surface in a non-ionic surfactant system using a time-resolved small-angle neutron-scattering technique. The cubic lattice transformed from a hexagonal phase showed a polycrystalline scattering pattern and strong intensity fluctuation as a function of time. A characteristic correlation time of the intensity fluctuation obtained by time correlation function analysis was about 150 min. In the case of the cubic lattice from a lamellar phase the diffraction peaks showed moncrystalline-like pattern and no intensity fluctuation. We consider that the fluctuations arise from the polycrystalline cubic network trapped in a metastable state. In other words, the cubic phase from the hexagonal phase is in an incubation time of finite-to-infinite transition of the cubic network.

Laser light scattering and small-angle neutron scattering were used to study poly(oxybutylene)−poly(oxyethylene)−poly(oxybutylene) triblock copolymers (B6E46B6) in aqueous solution from low to high concentrations and over a range of temperatures from 5 to 35 °C. B6E46B6 molecules exist as unimers at low concentrations and low temperatures. At higher concentrations and at low temperature (≤15 °C), they associate in small numbers and scattering evidence shows that molecular associates with open structures might form. At higher temperatures, typical flowerlike micelles form. The critical micelle concentration decreases with increasing temperature while the association number increases. At high polymer concentrations (e.g., at 35% in mass), further entanglements form among the micelles, yielding another slow mode in dynamic light scattering, which can be attributed to the bridging of two hydrophobic end blocks located in two different hydrophobic clusters by the hydrophilic middle block. However, these cross-...

The Pluronic polyol F127, PEO99PPO69PEO99 (PEO and PPO being poly(ethylene oxide) and poly(propylene oxide), respectively) has the potential to be used as an effective separation medium in capillary electrophoresis (CE) for the separation of biomacromolecules such as DNA fragments and proteins. Static light scattering (SLS), dynamic light scattering (DLS), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) were used to characterize the solution properties and the microstructures of F127 in the same electrophoresis buffer used in CE. In the dilute solution region, F127 in CE buffer forms micelles similar to that in water. At high solution concentrations, micelles tend to pack into some crystalline forms with relatively well-structured PPO centers. By using a combination of SANS and SAXS results, we are able to conclusively determine the gellike structure to have a face-centered cubic lattice. The effects of solvent, polymer concentration, temperature, and sample preparation proc...

We have investigated stability of a cubic lattice having a periodic-minimal surface in a nonionic surfactant system using a time resolved small-angle neutron scattering technique. The cubic lattice transformed from a lamellar phase showed polycrystalline scattering patterns and a strong fluctuation of the diffraction intensity as a function of time. The intensity distribution analysis indicates that the domain coarsening proceeds until 2000 min and then the coarsening is suppressed. At a critical temperature, such fluctuating peaks suddenly grow to a monocrystalline pattern showing no intensity fluctuations. On the contrary, in the case of the cubic lattice transformed from a hexagonal phase, the polycrystalline diffraction peaks also showed fluctuating behavior, but in this case, the domain coarsening is suppressed and the scattering intensity fluctuates around an equilibrium value. We consider that the fluctuations arise from the polycrystalline cubic network trapped in a metastable state.

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

International audience; We present the current status of development of the two macromolecular crystallography (MX) beamlines, FMX and AMX, and the X-ray scattering beamline LIX, at the National Synchrotron Light Source-II (NSLS-II) [1]. Together, FMX and AMX will cover a broad range of use cases from serial crystallography on micron sized crystals, to very large unit cell complexes, to rapid sample screening, e. g. for the always-hard-to-grow membrane proteins and for ligand binding studies. The LIX beamline will support a variety of X-ray scattering measurements for studies on proteins in solution, lipid membranes and biological tissues. We have performed Synchrotron Radiation Workshop (SRW) [2] and Shadow[3] simulations to help select optimal methods to modify the size of the beam easily and smoothly at both FMX and AMX. The very low emittance of the NSLS-II storage ring and the resulting low divergence of the X-ray beam, as well as the long optical path lengths in the photon delivery systems lead to stringent requirements e. g. for vibrational stability and mirror quality. We discuss beamline design considerations addressing these challenges, such as combining mirror optics with compound refractive lenses (CRLs)

The supramolecular structure, formed by a PEO-PPO-PEO copolymer, PEO 13 PEO 30 -PEO 13 or Pluronic L64, in mixtures of xylene/water, was investigated by small-angle X-ray scattering over a temperature range of 25–60°C and by small-angle neutron scattering over a temperature range of 13–29°C. The large aggregates had a hexagonally packed cylindrical structure with the interdomain spacing being in the order of ∼ 10 nm interdomain spacing increased with increasing amount of solubilized water in the large aggregates.

In order to elucidate the structural changes that occur during the hydrolysis of ATP by myosin, low-angle neutron and X-ray scattering have been used to investigate the shape of the myosin head (S1) with various bound nucleotides and nucleotide analogs. It was found that the radius of gyration (Rg) of S1.MgADP.AlF4 and of S1MgADP.Vi were similar and significantly smaller (approximately 3%) than the similar Rg values of nucleotide-free S1, S1.MgADP and S1.MgADP.BeFx. In addition, S1 in the presence of MgATP, which is predominantly in the S1.MgADP.Pi state under the experimental conditions employed, showed a change in Rg comparable with that of S1.MgADP.AlF4 and S1.MgADP.Vi. The results obtained here with BeFx and AlF4 are in close harmony with crystallographic results on truncated S1 bearing MgADP.BeFx and MgADP.AlF4. A. Fisher and co-workers have postulated that these two systems, which exhibit some structural differences, represent the pre-hydrolysis state and the transition state of ATP hydrolysis, respectively. It was postulated that this structural difference might alter the orientation of the light-chain-binding domain (tail) of intact S1 relative to the remainder of the molecule. Since this orientation is the major determinant of the Rg of S1, the current data support the hypothesis that a unitary large-scale conformational cocking of S1 for subsequent force production occurs just before or during ATP hydrolysis. Modeling changes in Rg by rigid-body rotations indicates that the longitudinal component of the force-producing throw is likely to be less than 6 nm.

Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size ( xi ) can be expressed by the relation xi = 2.09C-0.76, where xi is in A and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecules. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.

Small-angle neutron scattering (SANS) and rheometry were used to investigate the supramolecular structure formed by a poly(oxyethylene−oxypropylene−oxyethylene) (PEO100PPO65PEO100, Pluronic F127) copolymer in aqueous solution over a temperature range of 10−75 °C and a concentration range of 10−20 wt %. At copolymer concentrations of less than 12.5% the solutions are Newtonian fluids. Gels with an ordered structure (cubic packing of spherical micelles) are observed over a well-defined temperature window when the copolymer concentrations are greater than 17 wt %. The SANS results show that the aggregation number of the micelles is independent of temperature and concentration. Low-yield stresses, very high zero shear viscosities (∼106 P), and shear thinning are the major rheological characteristics of the gels. Near, but outside, the gel phase boundary the solutions are non-Newtonian (shear thinning). However, SANS shows these solutions contain domains having the same structure as that in the gel phase. The ...

Small-angle neutron scattering (SANS) was used to investigate the micellar structure formed by a poly(oxyethylene-oxypropylene-oxyethylene) (PEO{sub 13}PPO{sub 30}PEO{sub 13}, Pluronic L64) copolymer in D{sub 2}O over a temperature range of 8.4-35.0{degree}C. The intermicellar interactions were corrected by using an equivalent hard sphere approximation with an equivalent hard sphere radius. The aggregation number of the micelles decreased with decreasing temperature. The micellar scattering behavior could be well described by a core-shell structure. Based on the core-shell model, the volume fraction of the polymer segments in the micellar shell was less than 0.2. The micellar molar mass became larger after having introduced an organic solvent (xylene) into the system and increased with increasing amount of solubilized xylene. The maximum amount of solubilized xylene in the micelle was of the order of 0.3-0.4 xylene molecule per PO unit. 35 refs., 7 figs., 3 tabs.

The micellar structure is usually considered to be composed of a hard sphere (liquid) core and a heavily solvated corona. Therefore, the correction for intermicellar interactions at finite concentrations can be relatively complicated. In this article, small-angle neutron scattering of a copolymer, known as Pluronic L64 (PEO13PPO30PEO13), in o-xylene in the presence of D2O is used to demonstrate that, based on the hard sphere approximation, intermicellar interactions can be corrected by representing the micellar size as having an equivalent hard sphere radius. The procedure remains valid even if the micellar shape becomes asymmetric, with axial ratios of 3 ˜ 4. For the present system, the equivalent hard sphere radius corresponds to the micellar core radius plus one-half of the micellar shell thickness. With the equivalent hard sphere approach, the scattering behavior of the micelle could be described by using a core-shell structure. © 1994 John Wiley & Sons, Inc.

Polymeric micellar structures formed by a PEO-PPO-PEO copolymer in o-xylene in the presence of water were investigated by small-angle neutron scattering. In order to reveal the detailed micellar structure, different contrasts among the micellar core, the micellar shell, and the dispersing medium (background) were constructed by selectively changing the protonated/deuterated combination of water and xylene. The micellar structure could be well described by a core-shell structure with the scattering behavior of the micellar shell being very similar to that of a star polymer. The solubilized water existed not only in the micellar core but also in the micellar shell. The volume fraction of a copolymer segments in the micellar shell was rather low, being of the order of 0.2. There seemed to be no sharp interface between the micellar core and the micellar shell. 25 refs., 11 figs., 4 tabs.

We describe a concept for X-ray optics to feed a pair of macromolecular crystallography (MX) beamlines, which view canted undulator radiation sources in the same storage ring straight section. It can be deployed at NSLS-II and at other low-emittance third-generation synchrotron radiation sources where canted undulators are permitted, and makes the most of these sources and beamline floor space, even when the horizontal angle between the two canted undulator emissions is as little as 1–2 mrad. The concept adopts the beam-separation principles employed at the 23-ID (GM/CA-CAT) beamlines at the Advanced Photon Source (APS), wherein tandem horizontally deflecting mirrors separate one undulator beam from the other, following monochromatization by a double-crystal monochromator. The scheme described here would, in contrast, deliver the two tunable monochromatic undulator beams to separate endstations that address rather different and somewhat complementary purposes, with further beam conditioning imposed as required. A downstream micro-focusing beamline would employ dual-stage focusing for work at the micron scale and, unique to this design, switch to single-stage focusing for larger beams. On the other hand, the upstream, more highly automated beamline would only employ single-stage focusing.

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied. For free chymotrypsin-generated S1 it was found that Rg = 4.0 +/- 0.15 nm, while for papain-generated S1 it was found that Rg = 4.6 +/- 0.2 nm. Upon binding of papain-generated S1 to actin at low NS1/N actin ratios, the change in Rg in difference experiments was delta Rg = 0.05 +/- 0.15 nm. This lack of significant change in Rg in the very low-s domain confirms and extends our earlier neutron scattering work in the higher-s domain. The longest chords of S1, as well as shorter ones, are not significantly altered upon actin binding. These results indicate that muscle contraction does not occur as a result of large-scale changes in S1 structure. In actin-Tm complexes, a measurement of the mean cross-helix separation, d, of Tm molecules has been made using neutron scattering. With deuterated actin matched out in 93% D2O buffer, it was found that d = 7.9 +/- 0.3 nm. This value is in good agreement with a model based on Tm crystallography and also with recent electron microscopy results. These experiments demonstrate the feasibility and value of neutron diffraction and scattering techniques in the study of muscle contraction and its control. One can expect that the further employment of emerging cell biology techniques for generating deuterated proteins will aid our understanding of muscle in the future.

A high-flux insertion device and beamline for macromolecular crystallography has been built at the National Synchrotron Light Source (NSLS) that employs a mini-gap undulator source developed by the NSLS. The mini-gap undulator at beamline X29 is a hybrid-magnet device of period 12.5 mm operating at proven gaps of 3.3-10 mm. The beamline provides hard X-rays for macromolecular crystallography experiments from the second and third harmonics over an energy range of 5-15 keV. The X-ray optics is designed to deliver intense and highly collimated X-rays. Horizontal focusing is achieved by a cryogenically cooled sagittally focusing double-crystal monochromator with approximately 4.1:1 demagnification. A vertical focusing mirror downstream of the monochromator is used for harmonic rejection and vertical focusing. The experimental station hosts an Area Detector Systems Quantum 315 CCD detector with 2.2 s readout time between exposures and Crystal Logic goniostat for crystal rotation and detector positioning. An auto-mounter crystal changer has been installed to facilitate the high-throughput data collection required by the major users, which includes structural genomics projects and the Macromolecular Crystallography Research Resource mail-in program. X29 is 10(3) times brighter than any existing bending-magnet beamline at NSLS with an actual flux of 2.5 x 10(11) photons s(-1) through a 0.12 mm square aperture at 11.271 keV.

We present the current development status of the Advanced Beamlines for Biological Investigations with X-rays (ABBIX) at NSLS-II. This NIH-funded project includes an x-ray scattering beamline (LIX) and two macromolecular crystallography beamlines (FMX and AMX). User operations are scheduled to begin in 2016.Facilitating x-ray scattering studies on proteins in solution, lipid membranes and biological tissues, the High Brightness X-ray Scattering for Life Sciences beamline - LIX - will be equipped with a single long undulator (IVU23). Via a two-stage demagnification scheme it will produce beams down to a size of ∼1 µm, and up to several hundred microns. With a broad energy range of 2.1 - 18 keV (0.7 - 5.9 A) and capable of simultaneously collecting data on 3 detectors, it will support a variety of x-ray scattering measurements.In the neighboring sector are the pair of MX beamlines, equipped with two identical canted undulators (IVU21). The beamlines' specializations are complementary. The Frontier Microfocusing Macromolecular Crystallography - FMX - will deliver a high photon flux of 1013 ph/s at the Se K-edge into a spot of 1 µm width. It will cover a broad energy range from 5 - 30 keV, corresponding to wavelengths from 0.4 - 2.5 A. Beam sizes up to 50 µm will be available. The Highly Automated Macromolecular Crystallography - AMX - will be optimized for high throughput applications, with beam sizes from 4 - 100 µm and an energy range of 5 −18 keV (0.7 - 2.5 A). Together, FMX and AMX will cover a broad range of applications from serial crystallography on micron sized crystals, over very large unit cell complexes, to rapid sample screening, e.g. for ligand binding studies.

We report on the status of the development of three beamlines for the National Synchrotron Light Source-II (NSLS-II), two for macromolecular crystallography (MX), and one for wide- and small-angle x-ray scattering (SAXS). Funded by the National Institutes of Health, this suite of Advanced Beamlines for Biological Investigations with X-rays (ABBIX) is scheduled to begin operation by 2015. The two MX beamlines share a sector with identical canted in-vacuum undulators (IVU21). The microfocusing FMX beamline on the inboard branch employs a two-stage horizontal source demagnification scheme, will cover an energy range of 5 – 23 keV, and at 12.7 keV will focus a flux of up to 1013 ph/s into a spot of 1 μm width. The companion AMX beamline on the short outboard branch of the sector is tunable in the range of 5 – 18 keV and has a native focus of 4 μm (h) × 2 μm (v). This robust beamline will be highly automated, have high throughput capabilities, and with larger beams and low divergence will be well suited for structure determinations on large complexes. The high brightness SAXS beamline, LIX, will provide multiple dynamic and static experimental systems to support scientific programs in solution scattering, membrane structure determination, and tissue imaging. It will occupy a different sector, equipped with a single in-vacuum undulator (IVU23). It can produce beams as small as 1 μm across, and with a broad energy range of 2.1 – 18 keV it will support anomalous SAXS.

We have been engaged in studies of the structure and condensation of chromatin into the 30nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6–7 nucleosomes/11nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5–7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

The Escherichia coli ATP-dependent caseinolytic protease (Clp) is composed of two distinct subunits; protease, ClpP, and ATPase, ClpA. Active ClpP has been overexpressed to approximately 50% of soluble protein in E. coli, and purified to homogeneity. Direct mass determination of individual particles using scanning transmission electron microscopy (STEM) yields a mean native molecular mass of 305 +/- 9 kDa for the ClpP oligomer, suggesting that it has a tetradecameric structure. Small-angle X-ray scattering (SAXS) curves were determined for ClpP in solution at concentrations of 1-10 mg/mL. A combination of STEM and SAXS data was used to derive a model for ClpP, comprising a cylindrical oligomer about 100 A in diameter and about 75 A in height with an axial pore about 32-36 A in diameter. The volume of the pore is estimated to be approximately 70,000 A3, similar in size to those found in chaperone proteins, and is large enough to accommodate unfolded polypeptide chains, although most globular folded proteins would be excluded.

THE linker histone H1 binds to the nucleosome and is essential for the organization of nucleosomes into the 30-nm filament of chromatin1. It has been implicated in the repression of transcription2–5, and phosphorylation of H1 may be involved in cell-cycle-dependent chromatin condensation and decondensation6. A long-standing issue concerns the location of H1 in the chromatin filament7. The original solenoidal model8 proposes that H1 is inside the 30-nm filament, but other models, also helical, suggest a variable9 or more accessible10 location for H1. Investigations to determine the location of the linker histone based on its access-ibility to antibodies11–15 or immobilized proteases16 under various ionic conditions have yielded conflicting results. Here we use neutron scattering in a direct structural determination to show that H1 is located in the interior of the filament.

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered “invisible” to neutrons when dissolved in 94% D2O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometrics, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.

Lonny Berman,1 Marc Allaire,1 Mark Chance,3 Wayne Hendrickson,4 Annie Heroux,2 Babu Manjasetty,3 Allen Orville,2 Howard Robinson,2 Anand Saxena,2 Dieter Schneider,2 Wuxian Shi,3 Alexei Soares,2 Vivian Stojanoff,1 and Robert Sweet2 (1) Brookhaven National Laboratory, National Synchrotron Light Source, Bldg. 725D, Upton, New York 11973, USA (2) Brookhaven National Laboratory, Biology Dept., Bldg. 463, Upton, New York 11973, USA (3) Case Western Reserve University, Center for Synchrotron Biosciences, Cleveland, Ohio 44106, USA (4) Columbia University, Dept. of Biochemistry and Molecular Biophysics, New York, New York 10032, USA