Venugopalan Nagarajan

Argonne National Laboratory, Lemont, Illinois, United States

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Publications (7)31.01 Total impact

  • S. Xu, V. Nagarajan, R. F. Fischetti
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    ABSTRACT: Since 2007, we have offered single collimators for mini beams of sizes 5, 10 and 20 µm as well as a 300 µm scatter-guard to accommodate the fully focused beam. The advantages of varying the beam size were obvious, better signal/background ratio and the capability to raster with a coarse, larger beam, then fine tune with one of the mini-beam options. The mini beams proved to be a technical and popular success; however, the switching of the single collimators often involved staff intervention. The single mini-beam collimators were the precursors to the development of a triple collimator. This implementation incorporated two mini-beam collimators and a 300 µm scatter-guard on one post. The design was improved by consolidation of fabrication from a single piece of molybdenum block. It significantly improved the robustness, ease of initial alignment, reduction of background and increased automation. However, experimenters were still left with a choice of either a (5, 10 and 300 µm)- or a (10, 20 and 300 µm)-triple collimator. Recently, a quad collimator was developed and fabricated to provide a selection of mini beams of 5, 10 and 20 µm and a 300 µm scatter-guard, on a single post. We will present the mechanical design of multi-collimators, results of measured beam flux through the collimator pinholes.
    Diamond Light Source Proceedings. 10/2011; 1(MEDSI-6).
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    ABSTRACT: In October 2009, a quad, mini-beam collimator was implemented at GM/CA CAT that allowed users to select between a 5, 10, or 20 micron mini-beam or a 300 micron scatter guard for macromolecular crystallography. Initial alignment of each pinhole to the optical axis of each path through the mini-beam collimator is performed under an optical microscope using an alignment jig. Next, the pre-aligned collimator and its kinematic mount are moved to the beamline and attached to a pair of high precision translation stages attached to an on-axis-visualization system for viewing the protein crystal under investigation. The collimator is aligned to the beam axis by two angular and two translational motions. The pitch and yaw adjustments are typically only done during initial installation, and therefore are not motorized. The horizontal and vertical positions are adjusted remotely with high precision translational stages. Final alignment of the collimator is achieved using several endstation components, namely, a YAG crystal at the sample position to visualize the mini-beam, a CCD detector to record an X-ray background image, and a PIN diode to record the mini-beam intensity. The alignment protocol and its opto-mechanical instrumentation design will be discussed in detail.
    Proc SPIE 09/2011;
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    ABSTRACT: Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.
    Proceedings of the National Academy of Sciences 03/2011; 108(15):6127-32. · 9.81 Impact Factor
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    ABSTRACT: Recently, several important structures have been solved using micro-crystallographic techniques that previously could not have been solved with conventional crystallography. At GM/CA-CAT we continue to develop micro-crystallographic capabilities for difficult problems such as small crystals of large macromolecular complexes or membrane proteins grown in the lipidic cubic phase. This paper will describe three major upgrades to our arsenal of tools, ``mini-beam'' collimators, active beamstop, and an improved goniostat. Our ``mini-beam'' collimators have evolved to a new triple-collimator fabricated from molybdenum as a uni-body. This has significantly improved the robustness, ease of initial alignment, and reduction of background. More recently, two prototypes of a quad-collimator have been developed and fabricated to provide a selection of mini-beams of 5, 10, 20 mum and a 300 mum scatter-guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet these challenges we have designed and implemented a goniostat with a 1-micron peak-to-peak sphere of confusion. This is a significant improvement over the previous 6 micron sphere of confusion of the commercially available air-bearing and XY stages. Finally, an ``active beamstop'' has been constructed. This will provide non-invasive, real time feedback at the sample during data collection.
    AIP Conference Proceedings. 01/2010; 1234.
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    ABSTRACT: Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 microm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.
    Journal of The Royal Society Interface 07/2009; 6 Suppl 5:S587-97. · 4.91 Impact Factor
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    ABSTRACT: The high-brilliance X-ray beams from undulator sources at third-generation synchrotron facilities are excellent tools for solving crystal structures of important and challenging biological macromolecules and complexes. However, many of the most important structural targets yield crystals that are too small or too inhomogeneous for a ;standard' beam from an undulator source, approximately 25-50 microm (FWHM) in the vertical and 50-100 microm in the horizontal direction. Although many synchrotron facilities have microfocus beamlines for other applications, this capability for macromolecular crystallography was pioneered at ID-13 of the ESRF. The National Institute of General Medical Sciences and National Cancer Institute Collaborative Access Team (GM/CA-CAT) dual canted undulator beamlines at the APS deliver high-intensity focused beams with a minimum focal size of 20 microm x 65 microm at the sample position. To meet growing user demand for beams to study samples of 10 microm or less, a ;mini-beam' apparatus was developed that conditions the focused beam to either 5 microm or 10 microm (FWHM) diameter with high intensity. The mini-beam has a symmetric Gaussian shape in both the horizontal and vertical directions, and reduces the vertical divergence of the focused beam by 25%. Significant reduction in background was achieved by implementation of both forward- and back-scatter guards. A unique triple-collimator apparatus, which has been in routine use on both undulator beamlines since February 2008, allows users to rapidly interchange the focused beam and conditioned mini-beams of two sizes with a single mouse click. The device and the beam are stable over many hours of routine operation. The rapid-exchange capability has greatly facilitated sample screening and resulted in several structures that could not have been obtained with the larger focused beam.
    Journal of Synchrotron Radiation 04/2009; 16(Pt 2):217-25. · 2.19 Impact Factor
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    ABSTRACT: A simple apparatus for achieving beam sizes in the range 5-10 μm on a synchrotron beamline was implemented in combination with a small 125 x 25 μm focus. The resulting beam had sufficient flux for crystallographic data collection from samples smaller than 10 x 10 x 10 μm. Sample data were collected representing three different scenarios: (i) a complete 2.0 data set from a single strongly diffracting microcrystal, (ii) a complete and redundant 1.94 A data set obtained by merging data from six microcrystals and (iii) a complete 2.24 A data set from a needle-shaped crystal with less than 12 x 10 μm cross-section and average diffracting power. The resulting data were of high quality, leading to well refined structures with good electron-density maps. The signal-to-noise ratios for data collected from small crystals with the mini-beam were significantly higher than for equivalent data collected from the same crystal with a 125 x 25 μm beam. Relative to this large beam, use of the mini-beam also resulted in lower refined crystal mosaicities. The mini-beam proved to be advantageous for inhomogeneous large crystals, where better ordered regions could be selected by the smaller beam.
    Acta Crystallographica Section D Biological Crystallography 05/2008; 64(Pt 4):425-35. · 14.10 Impact Factor