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.

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.

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.

The first spectroscopic model for the S1 state multiline signal of the OEC.

The parallel-mode electron paramagnetic resonance (EPR) spectrum of the S(1) state of the oxygen-evolving complex (OEC) shows a multiline signal centered around g=12, indicating an integer spin system. The series of [Mn(2)(2-OHsalpn)(2)] complexes were structurally characterized in four oxidation levels (Mn(II)(2), Mn(II)Mn(III), Mn(III)(2), and Mn(III)Mn(IV)). By using bulk electrolysis, the [Mn(III)Mn(IV)(2-OHsalpn)(2)(OH)] is oxidized to a species that contains Mn(IV) oxidation state as detected by X-ray absorption near edge spectroscopy (XANES) and that can be formulated as Mn(IV)(4) tetramer. The parallel-mode EPR spectrum of this multinuclear Mn(IV)(4) complex shows 18 well-resolved hyperfine lines center around g=11 with an average hyperfine splitting of 36 G. This EPR spectrum is very similar to that found in the S(1) state of the OEC. This is the first synthetic manganese model complex that shows an S(1)-like multiline spectrum in parallel-mode EPR.

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.

Mini-beam collimator enables microcrystallography experiments on standard beamlines.

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.

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

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.

Models for the lower S states of photosystem II: a trinuclear mixed-valent MnII/MnIV/MnII complex.

The trinuclear complex Mn(3)(pko)(4)(CH(3)O)(2)(SCN)(2).CH(3)OH, 1, where Hpko is 2,2'-dipyridylketonoxime, is a rare example of a complex simultaneously containing Mn(II) and Mn(IV). X-ray crystallography and XANES spectroscopy clearly distinguish the Mn(II)(2)Mn(IV) valence isomer from the more commonly observed Mn(III)(2)Mn(II) formulation. Fits to variable-temperature magnetic susceptibility data indicate that the Mn(II) and Mn(IV) are ferromagnetically coupled (J = +6.13 cm(-1)) and that 1 has an S = (13)/(2) ground state.