Derek W. Yoder

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.

A 7 µm mini-beam improves diffraction data from small or imperfect crystals of macromolecules

An X-ray mini-beam of 8 × 6 µm cross-section was used to collect diffraction data from protein microcrystals with volumes as small as 150–300 µm3. The benefits of the mini-beam for experiments with small crystals and with large inhomogeneous crystals are investigated.

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.

Fast fluorescence techniques for crystallography beamlines.

This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples.

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...

One‐Micron Beams for Macromolecular Crystallography at GM/CA‐CAT

GM/CA‐CAT has developed a 1‐μm beam for challenging micro‐diffraction experiments with macromolecular crystals (e.g. small crystals) and for radiation damage studies. Reflective (Kirkpatrick‐Baez mirrors) and diffractive (Fresnel zone plates) optics have been used to focus the beam. Both cases are constrained by the need to maintain a small beam convergence. Using two different zone plates, 1.0×1.0 and 0.8×0.9 μm2 (V×H,FWHM) beams were created at 15.2 keV and 18.5 keV, respectively. Additionally, by introducing a vertical focusing mirror upstream of the zone plate, a line focus at 15.2 keV was created (28×1.4 μm2 V×H,FWHM) with the line oriented perpendicular to the X‐ray polarization and the crystal rotation axis. Crystal‐mounting stages with nanometer resolution have been assembled to profile these beams and to perform diffraction experiments.

Optical Performance of the GM/CA‐CAT Canted Undulator Beam lines for Protein Crystallography

A new macromolecular crystallographic facility developed by GM/CA‐CAT is operational at the Advanced Photon Source (APS). The facility consists of three beamlines: two lines based on the first “hard” dual canted undulators and one bending magnet beamline. The ID lines are operational, and the BM line is being commissioned. Both insertion device (ID) beamlines are independently tunable over a wide energy range. The inboard ID lines have been upgraded with a new insertion device to provide enhanced performance for MAD phasing experiments near the selenium and bromine K‐edges. The ID line monochromators’ crystals are indirectly, cryogenically cooled for improved performance and reliability. Focusing is achieved by long bimorph mirrors in a Kirkpatrick‐Baez geometry. This paper describes the design of the beam lines and the optical characterization of the mirrors and monochromators.

Predicted optical performance of the GM/CA@APS micro-focus beamline

GM/CA at the APS has developed microcrystallography capabilities for structural biology applications. The robust, quad, mini-beam collimators, which enable users to rapidly select between a 5, 10 or 20 micron diameter beam or a scatter guard for the full focused beam, are coupled with several powerful automated software tools that are built into the beamline control system JBluIce-EPICS. Recent successes at beamlines around the world in solving structures from microcrystals (2 - 10 microns) have led to increased demand for high-intensity micro-focus beams. We have designed a new micro-focus endstation to increase the intensity in mini- and micro-beams at GM/CA by one to two orders of magnitude to meet this growing demand. The new optical design is based on the well-established approach of using two-stage demagnification. The existing bimorph mirrors, arranged in a Kirkpatrick-Baez geometry, focus the beam onto slits located upstream of the sample whereby the slit aperture defines a secondary source, that is reimaged with a second pair of mirrors. This design incorporates two focal modes: a mini-beam mode where the beam is focused to 20-micron diameter and a micro-beam mode where it is focused to 5-microns. The size of the secondary source aperture can be varied rapidly (seconds) to adjust the beam size at the sample position in two ranges 20 - 3 micron and 5 - 1 micron. The second set of mirrors will each have two super polished ellipses allowing quick (minutes) interchange between modes.

Simulation and optimization of a sub-micron beam for macromolecular crystallography using SHADOW and XOP at GM/CA CAT at the APS

The small, high intensity and low convergence beams available on beamlines at 3rd generation synchrotron sources have been a boon to macromolecular crystallography. It is now becoming routine to solve structures using a beam in the 5 - 20 micron (FWHM) range. However, many problems in structural biology suffer from poor S/N due to small (a few microns) crystals or larger inhomogenous crystals. In additional, theoretical calculations and experimental results have demonstrated that radiation damage may be reduced by using a micron-sized X-ray beam. At GM/CA CAT we are developing a sub-micron, low convergence beam to address these issues. The sub-micron beam capability will be developed on the existing beamline 23ID-D where the minimum beam size available to users is currently 5 microns in diameter. The target goals are a beam size of ~0.8 micron (FWHM) in diameter, with a beam convergence of less 0.6 milli-rads, a flux greater than 5×1010 photons/sec, and an energy range from 5 to 35 keV. Five optical systems will be compared: 1) a single set of highly demagnifying Kirkpatrick-Baez (K-B) mirrors, 2) multiple Fresnel Zone Plates (FZP), 3) a set of K-B mirrors focusing to a secondary source that is imaged by another set of K-B mirrors, 4) a set of K-B mirrors focusing to a secondary source that is imaged by a FZP, 5) a horizontal focusing mirror focusing to a secondary source that is imaged by another horizontal mirror together with a vertical focusing mirror. Here we will present the results of a design optimization based on ray trace simulations (SHADOW), flux calculations (XOP), and experimental results on 23ID.

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.