Sudhir Babu Pothineni

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

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.

JBluIce–EPICS control system for macromolecular crystallography

The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

Automated detection and centring of cryocooled protein crystals.

A novel method is presented for the automated recognition of cryocooled macromolecular crystals. The method uses several texture-based image-processing algorithms for automated crystal centring, which are able to cope with a variety of crystal morphologies and illumination conditions. The results combined from different algorithms, together with their estimated standard uncertainties, provide a robust determination of the crystal location and allow an internal assessment of their reliability. The method was coded within the software XREC and showed a good performance on 104 sets of images from various beamlines.

Micro‐Crystallography Developments at GM/CA‐CAT at the APS

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 μm and a 300 μm scatter‐guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet t...

Tightly integrated single- and multi-crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system

Single- and multi-crystal data collection strategy and automated data processing have been tightly integrated into the JBluIce graphical user interface. Grid Engine is used to distribute these processes into multiple workstations to make efficient use of all available computing resources.

Remote Access Capabilities at the GM/CA Beamlines at the APS

The GM/CA facility consists of two undulator source beamlines and a bending magnet beamline at the Advanced Photon Source (APS). Access to the operation of these beamlines is accomplished through visits by investigators who are either on-site, remote or a combination of the two. In all modes of access, user operations are controlled by the experimenter. The control and capabilities of the GM/CA beamlines are identical for remote and on-site users. Remote access to the beamlines is through NX or Teamviewer to local computers [1]. Once communication has been established, experienced GM/CA experimenters are greeted by our familiar JBluIce, the graphical user interface/control program[2] responsible for all operations from sample handling through data collection and reduction. Although investigators always see a familiar interface, both software and hardware on the beamlines are continually improving. Recent hardware upgrades include a shift of the optical focusing mirrors on the ID-B beamline closer to the sample to provide a significant increase in flux, and installation of a new Pilatus3 6M detector on ID-D, the second undulator beamline. The JBluIce program has incorporated new detector controls for shutterless operation while continuing to expand the features of rastering, vector (helical) data collection, strategy tools and data analysis. These tools have been essential to investigators working on membrane crystal samples, e.g. GPCRs, as well as for samples that decay quickly or require data to be collected from multiple crystals. The presentation will provide an overview of beamline remote control as well as an update of the equipment that it operates at GM/CA.

JBluIce-EPICS: A fast and flexible open-source beamline control system for macromolecular crystallography

This paper overviews recent advances in the JBluIce-EPICS open-source control system designed at the macromolecular crystallography beamlines of the National Institute of General Medical Sciences and National Cancer Institute at the Advanced Photon Source (GM/CA@APS). We discuss some technical highlights of this system distinguishing it from the competition, such as reduction of software layers to only two, possibility to operate JBluIce in parallel with other beamline controls, plugin-enabled architecture where the plugins can be written in any programming language, and utilization of the whole power of the Java integrated development environment in the Graphical User Interface. Then, we demonstrate how these highlights help to make JBluIce fast, easily adaptable to new beamline developments, and intuitive for users. In particular, we discuss several recent additions to the system including a bridge between crystal rastering and data collection, automatic detection of raster polygons from optical crystal centering, background data processing, and a pathway to a fully automated pipeline from crystal screening to solving crystal structure.

JBluIce-EPICSbeamline control system for macromolecular crystallography

Carbon nanotubes are nanometer sized channels that have a very high potential for applications such as selective chemical filtration or water desalination. For such purposes, large membranes of parallel nanotubes, where all metal-based catalyst particles potentially obstructing nanotubes have been removed, are needed. An original experimental setup based on X-ray scattering and fluorescence has been designed to characterize membranes of arbitrarily large surface (cm2 to m2) [1]. We are able to determine quantitatively the nanotube alignment in the membrane, the density of nanotubes and to check for the removal of metal-based nanoparticles. We will present the set-up, the models we developed to analyze experimental data and some results obtained on the set-up. The set-up/modeling can also be adapted to others membranes than those of nanotubes, using small or wide angle scattering depending on the membrane composition. It should be underlined than although the analysis requires complete orientational modeling within the framework of X-ray scattering theory, the set-up is designed for a non-specialist and could be used for instance on an industrial production line, automatic fitting of the data providing one with two-dimensional mappings of the density, chemical composition and nanotube orientation in direct space (figure).