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Woodward, United States

Huang R.,Hauptman Woodward Institute | Meron M.,University of Chicago | Kujala N.,Illinois Institute of Technology | Barrea R.A.,Illinois Institute of Technology
Journal of Synchrotron Radiation | Year: 2010

Micro-focusing optical devices at synchrotron beamlines usually have a limited acceptance, but more flux can be intercepted if such optics are used to focus secondary sources created by the primary optics. Flux throughput can be maximized by placing the secondary focusing optics close to or exactly at the secondary source position. However, standard methods of beamline optics analysis, such as the lens equation or matching the mirror surface to an ellipse, work poorly when the source-to-optics distance is very short. In this paper the general characteristics of the focusing of beams with Gaussian profiles by a thin lens are analysed under the paraxial approximation in phase space, concluding that the focusing of a beam with a short source-to-optics distance is distinct from imaging the source; slope errors are successfully included in all the formulas so that they can be used to calculate beamline focusing with good accuracy. A method is also introduced to use the thin-lens result to analyse the micro-focusing produced by an elliptically bent trapezoid-shaped Kirkpatrick-Baez mirror. The results of this analysis are in good agreement with ray-tracing simulations and are confirmed by the experimental results of the secondary focusing at the 18-ID Bio-CAT beamline (at the APS). The result of secondary focusing carried out at 18-ID using a single-bounce capillary can also be explained using this phase-space analysis. A discussion of the secondary focusing results is presented at the end of this paper. © 2010 International Union of Crystallography.

Huang R.,Hauptman Woodward Institute | Meron M.,University of Chicago
Nuclear Instruments and Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment | Year: 2013

The techniques for the cooling of the first crystal of a monochromator are by now mature and are used routinely to deal with the heat loads resulting from the intense beams generated by third generation synchrotron insertion device sources. However, the thermal stability of said monochromators, which crucially depends on proper shielding of X-ray scattering off the first crystal, remains a serious consideration. This will become even more so in the near future, as many synchrotron facilities are upgrading to higher beam currents and energies. During a recent upgrade of the 17-ID beamline at the APS it was recognized that accurate simulation of the spatial distribution of the power scattered off the first crystal was essential for the understanding and remediation of the observed large temperature increase of the first crystal's scattering shield. The calculation is complex, due to the broad energy spectrum of the undulator and the prevalence of multiple X-ray scattering events within the bulk of the crystal, thus the Monte Carlo method is the natural tool for such a task. A successful simulation was developed, for the purpose of the 17-ID upgrade, and used to significantly improve the design of the first crystal's scattering shield. © 2013 Elsevier B.V.

Huang R.,Hauptman Woodward Institute | Huang R.,Cornell University
Journal of Synchrotron Radiation | Year: 2011

One of the challenges of tuning bimorph mirrors with many electrodes is that the calculated focusing voltages can be different by more than the safety limit (such as 500 V for the mirrors used at 17-ID at the Advanced Photon Source) between adjacent electrodes. A study of this problem at 17-ID revealed that the inverse problem of the tuning in situ, using X-rays, became ill-conditioned when the number of electrodes was large and the calculated focusing voltages were contaminated with measurement errors. Increasing the number of beamlets during the tuning could reduce the matrix condition number in the problem, but obtaining voltages with variation below the safety limit was still not always guaranteed and multiple iterations of tuning were often required. Applying Tikhonov regularization and using the L-curve criterion for the determination of the regularization parameter made it straightforward to obtain focusing voltages with well behaved variations. Some characteristics of the tuning results obtained using Tikhonov regularization are given in this paper. © 2011 International Union of Crystallography Printed in Singapore - all rights reserved.

Clark K.M.,University of Rochester | Fedoriw N.,University of Rochester | Robinson K.,University of Rochester | Connelly S.M.,University of Rochester | And 4 more authors.
Protein Expression and Purification | Year: 2010

To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents. © 2009 Elsevier Inc. All rights reserved.

Qian S.,Oak Ridge National Laboratory | Dean R.,Hauptman Woodward Institute | Dean R.,State University of New York at Buffalo | Urban V.S.,Oak Ridge National Laboratory | And 3 more authors.
PLoS ONE | Year: 2012

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A "DNA outside, protein inside" mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.

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