MxDC and MxLIVE: software for data acquisition,information management and remote access to macromolecular crystallography beamlines

An integrated computer software system for on‐site and remote collection of macromolecular crystallography (MX) data at the Canadian Light Source (CLS) is described. The system consists of an integrated graphical user interface for data collection and beamline control [MX Data Collector (MxDC)] which provides experiment‐focused control of beamline devices, and a laboratory information management system [MX Laboratory Information Virtual Environment (MxLIVE)] for managing sample and experiment information through a web browser. The system allows remote planning and transmission of sample and experiment parameters to the beamline through MxLIVE, on‐site or remote data collection through MxDC guided by information from MxLIVE, and remote monitoring and download of experimental results through MxLIVE. The system is deployed and in use on both MX beamlines at the CLS which constitute the Canadian Macromolecular Crystallography Facility.     

BioCARS: a synchrotron resource for time‐resolved X‐ray science

BioCARS, a NIH‐supported national user facility for macromolecular time‐resolved X‐ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator‐based beamline optimized for single‐shot laser‐pump X‐ray‐probe measurements with time resolution as short as 100 ps. The source consists of two in‐line undulators with periods of 23 and 27 mm that together provide high‐flux pink‐beam capability at 12 keV as well as first‐harmonic coverage from 6.8 to 19 keV. A high‐heat‐load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X‐ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high‐speed chopper isolates single X‐ray pulses at 1 kHz in both hybrid and 24‐bunch modes of the APS storage ring. In hybrid mode each isolated X‐ray pulse delivers up to ～4 × 10¹⁰ photons to the sample, thereby achieving a time‐averaged flux approaching that of fourth‐generation X‐FEL sources. A new high‐power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage‐ring RF clock with long‐term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.     

A new optical scheme for large‐extraction small‐aberration vacuum‐ultraviolet synchrotron radiation beamlines

Vacuum‐ultraviolet radiation delivered by bending‐magnet sources is used at numerous synchrotron radiation facilities worldwide. As bending‐magnet radiation is inherently much less collimated compared with undulator sources, the generation of high‐quality intense bending‐magnet vacuum‐ultraviolet photon beams is extremely demanding in terms of the optical layout due to the necessary larger collection apertures. In this article, an optimized optical layout which takes into account both the optical and electron beam properties is proposed. This layout delivers an improved beam emittance of over one order of magnitude compared with existing vacuum‐ultraviolet bending‐magnet beamlines that, up to now, do not take into account electron beam effects. The arrangement is made of two dedicated mirrors, a cylindrical and a cone‐shaped one, that focus independently both the horizontal and the vertical emission of a bending‐magnet source, respectively, and has been already successfully applied in the construction of the infrared beamline at the Brazilian synchrotron. Using this scheme, two vacuum‐ultraviolet beamline designs based on a SOLEIL synchrotron bending‐magnet source are proposed and analysed. They would be useful for future upgrades to the DISCO beamline at SOLEIL and could be readily implemented at other synchrotron radiation facilities.     

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, ～25–50 µm (FWHM) in the vertical and 50–100 µm 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 µm × 65 µm at the sample position. To meet growing user demand for beams to study samples of 10 µm or less, a `mini‐beam' apparatus was developed that conditions the focused beam to either 5 µm or 10 µm (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.     

Automated sample‐scanning methods for radiation damage mitigation and diffraction‐based centering of macromolecular crystals

Automated scanning capabilities have been added to the data acquisition software, JBluIce‐EPICS, at the National Institute of General Medical Sciences and the National Cancer Institute Collaborative Access Team (GM/CA CAT) at the Advanced Photon Source. A `raster' feature enables sample centering via diffraction scanning over two‐dimensional grids of simple rectangular or complex polygonal shape. The feature is used to locate crystals that are optically invisible owing to their small size or are visually obfuscated owing to properties of the sample mount. The raster feature is also used to identify the best‐diffracting regions of large inhomogeneous crystals. Low‐dose diffraction images taken at grid positions are automatically processed in real time to provide a quick quality ranking of potential data‐collection sites. A `vector collect' feature mitigates the effects of radiation damage by scanning the sample along a user‐defined three‐dimensional vector during data collection to maximize the use of the crystal volume and the quality of the collected data. These features are integrated into the JBluIce‐EPICS data acquisition software developed at GM/CA CAT where they are used in combination with a robust mini‐beam of rapidly changeable diameter from 5 µm to 20 µm. The powerful software–hardware combination is being applied to challenging problems in structural biology.     

Protein crystallography beamline (PX‐BL21) at Indus‐2 synchrotron

The protein crystallography beamline (PX‐BL21), installed at the 1.5 T bending‐magnet port at the Indian synchrotron (Indus‐2), is now available to users. The beamline can be used for X‐ray diffraction measurements on a single crystal of macromolecules such as proteins, nucleic acids and their complexes. PX‐BL21 has a working energy range of 5–20 keV for accessing the absorption edges of heavy elements commonly used for phasing. A double‐crystal monochromator [Si(111) and Si(220)] and a pair of rhodium‐coated X‐ray mirrors are used for beam monochromatization and manipulation, respectively. This beamline is equipped with a single‐axis goniometer, Rayonix MX225 CCD detector, fluorescence detector, cryogenic sample cooler and automated sample changer. Additional user facilities include a workstation for on‐site data processing and a biochemistry laboratory for sample preparation. In this article the beamline, other facilities and some recent scientific results are briefly described.     

Advantages of a synchrotron bending magnet as the sample illuminator for a wide‐field X‐ray microscope

In this paper the choice between bending magnets and insertion devices as sample illuminators for a hard X‐ray full‐field microscope is investigated. An optimized bending‐magnet beamline design is presented. Its imaging speed is very competitive with the performance of similar microscopes installed currently at insertion‐device beamlines. The fact that imaging X‐ray microscopes can accept a large phase space makes them very well suited to the output characteristics of bending magnets which are often a plentiful and paid‐for resource. There exist opportunities at all synchrotron light sources to take advantage of this finding to build bending‐magnet beamlines that are dedicated to transmission X‐ray microscope facilities. It is expected that demand for such facilities will increase as three‐dimensional tomography becomes routine and advanced techniques such as mosaic tomography and XANES tomography (taking three‐dimensional tomograms at different energies to highlight elemental and chemical differences) become more widespread.     

Acoustic methods for high‐throughput protein crystal mounting at next‐generation macromolecular crystallographic beamlines

To take full advantage of advanced data collection techniques and high beam flux at next‐generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and align many samples per second. One approach is to use an acoustic ejector to eject crystal‐containing droplets onto a solid X‐ray transparent surface, which can then be positioned and rotated for data collection. Proof‐of‐concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.     

08B1‐1: an automated beamline for macromolecular crystallography experiments at the Canadian Light Source

Beamline 08B1‐1 is a recently commissioned bending‐magnet beamline at the Canadian Light Source. The beamline is designed for automation and remote access. Together with the undulator‐based beamline 08ID‐1, they constitute the Canadian Macromolecular Crystallography Facility. This paper describes the design, specifications, hardware and software of beamline 08B1‐1. A few scientific results using data obtained at the beamline will be highlighted.     

Beamline AR‐NW12A: high‐throughput beamline for macromolecular crystallography at the Photon Factory

AR‐NW12A is an in‐vacuum undulator beamline optimized for high‐throughput macromolecular crystallography experiments as one of the five macromolecular crystallography (MX) beamlines at the Photon Factory. This report provides details of the beamline design, covering its optical specifications, hardware set‐up, control software, and the latest developments for MX experiments. The experimental environment presents state‐of‐the‐art instrumentation for high‐throughput projects with a high‐precision goniometer with an adaptable goniometer head, and a UV‐light sample visualization system. Combined with an efficient automounting robot modified from the SSRL SAM system, a remote control system enables fully automated and remote‐access X‐ray diffraction experiments.     

DISCO: a low‐energy multipurpose beamline at synchrotron SOLEIL

DISCO, a novel low‐energy beamline covering the spectrum range from the VUV to the visible, has received its first photons at the French synchrotron SOLEIL. In this article the DISCO design and concept of three experimental stations serving research communities in biology and chemistry are described. Emphasis has been put on high flux generation and preservation of polarization at variable energy resolutions. The three experiments include a completely new approach for microscopy and atmospheric pressure experiments as well as a `classical' synchrotron radiation circular dichroism station. Preliminary tests of the optical design and technical concept have been made. Theoretical predictions of the beam have been compared with the first images produced by the first photons originating from the large‐aperture bending‐magnet source. Results are also reported concerning the cold finger used to absorb hard X‐ray radiation in the central part of the synchrotron beam and to avoid heavy thermal load on the following optics. Wavelength selection using monochromators with different gratings for each experimental set‐up as well as beam propagation and conditioning throughout the optical system are detailed. First photons comply very well with the theoretical calculations.     

ID29: a high‐intensity highly automated ESRF beamline for macromolecular crystallography experiments exploiting anomalous scattering

ID29 is an ESRF undulator beamline with a routinely accessible energy range of between 20.0 keV and 6.0 keV (λ = 0.62 Å to 2.07 Å) dedicated to the use of anomalous dispersion techniques in macromolecular crystallography. Since the beamline was first commissioned in 2001, ID29 has, in order to provide an improved service to both its academic and proprietary users, been the subject of almost continuous upgrade and refurbishment. It is now also the home to the ESRF Cryobench facility, ID29S. Here, the current status of the beamline is described and plans for its future are briefly outlined.     

Characterization of wax as a potential diffraction intensity standard for macromolecular crystallography beamlines

A number of commercially available waxes in the form of thin disc samples have been investigated as possible diffraction intensity standards for macromolecular crystallography synchrotron beamlines. Synchrotron X‐ray powder diffraction measurements show that beeswax offers the best performance of these waxes owing to its polycrystallinity. Crystallographic lattice parameters and diffraction intensities were examined between 281 and 309 K, and show stable and predictable thermal behaviour. Using an X‐ray beam of known incident flux at λ = 1 Å, the diffraction power of two strong Bragg reflections for beeswax were quantified as a function of sample thickness and normalized to 10¹⁰ photons s^?1. To demonstrate its feasibility as a diffraction intensity standard, test measurements were then performed on a new third‐generation macromolecular crystallography synchrotron beamline.     

XDS: a flexible beamline for X‐ray diffraction and spectroscopy at the Brazilian synchrotron

The majority of the beamlines at the Brazilian Synchrotron Light Source Laboratory (LNLS) use radiation produced in the storage‐ring bending magnets and are therefore currently limited in the flux that can be used in the harder part of the X‐ray spectrum (above ～10 keV). A 4 T superconducting multipolar wiggler (SCW) was recently installed at LNLS in order to improve the photon flux above 10 keV and fulfill the demands set by the materials science community. A new multi‐purpose beamline was then installed at the LNLS using the SCW as a photon source. The XDS is a flexible beamline operating in the energy range between 5 and 30 keV, designed to perform experiments using absorption, diffraction and scattering techniques. Most of the work performed at the XDS beamline concentrates on X‐ray absorption spectroscopy at energies above 18 keV and high‐resolution diffraction experiments. More recently, new setups and photon‐hungry experiments such as total X‐ray scattering, X‐ray diffraction under high pressures, resonant X‐ray emission spectroscopy, among others, have started to become routine at XDS. Here, the XDS beamline characteristics, performance and a few new experimental possibilities are described.     

HAXPES beamline PES‐BL14 at the Indus‐2 synchrotron radiation source

The Hard X‐ray Photo‐Electron Spectroscopy (HAXPES) beamline (PES‐BL14), installed at the 1.5 T bending‐magnet port at the Indian synchrotron (Indus‐2), is now available to users. The beamline can be used for X‐ray photo‐emission electron spectroscopy measurements on solid samples. The PES beamline has an excitation energy range from 3 keV to 15 keV for increased bulk sensitivity. An in‐house‐developed double‐crystal monochromator [Si (111)] and a platinum‐coated X‐ray mirror are used for the beam monochromatization and manipulation, respectively. This beamline is equipped with a high‐energy (up to 15 keV) high‐resolution (meV) hemispherical analyzer with a microchannel plate and CCD detector system with SpecsLab Prodigy and CasaXPS software. Additional user facilities include a thin‐film laboratory for sample preparation and a workstation for on‐site data processing. In this article, the design details of the beamline, other facilities and some recent scientific results are described.     

Carbon contamination of soft X‐ray beamlines: dramatic anti‐reflection coating effects observed in the 1 keV photon energy region

Carbon contamination is a general problem of under‐vacuum optics submitted to high fluence. In soft X‐ray beamlines carbon deposit on optics is known to absorb and scatter radiation close to the C K‐edge (280 eV), forbidding effective measurements in this spectral region. Here the observation of strong reflectivity losses is reported related to carbon deposition at much higher energies around 1000 eV, where carbon absorptivity is small. It is shown that the observed effect can be modelled as a destructive interference from a homogeneous carbon thin film.     

Understanding the instrumental profile of synchrotron radiation X‐ray powder diffraction beamlines

A Monte Carlo algorithm has been developed to calculate the instrumental profile function of a powder diffraction synchrotron beamline. Realistic models of all optical elements are implemented in a ray‐tracing software. The proposed approach and the emerging paradigm have been investigated and verified for several existing X‐ray powder diffraction beamlines. The results, which can be extended to further facilities, show a new and general way of assessing the contribution of instrumental broadening to synchrotron radiation data, based on ab initio simulations.     

Room‐temperature scavengers for macromolecular crystallography: increased lifetimes and modified dose dependence of the intensity decay

The advent of highly intense wiggler and undulator beamlines has reintroduced the problem of X‐ray radiation damage in protein crystals even at cryogenic temperatures (100 K). Although cryocrystallography can be utilized for the majority of protein crystals, certain macromolecular crystals (e.g. of viruses) suffer large increases in mosaicity upon flash cooling and data are still collected at room temperature (293 K). An alternative mechanism to cryocooling for prolonging crystal lifetime is the use of radioprotectants. These compounds are able to scavenge the free radical species formed upon X‐ray irradiation which are thought to be responsible for part of the observed damage. Three putative radioprotectants, ascorbate, 1,4‐benzoquinone and 2,2,6,6‐tetramethyl‐4‐piperidone (TEMP), were tested for their ability to prolong lysozyme crystal lifetimes at 293 K. Plots of relative summed intensity against dose were used as a metric to assess radioprotectant ability: ascorbate and 1,4‐benzoquinone appear to be effective, whereas studies on TEMP were inconclusive. Ascorbate, which scavenges OH radicals (k_OH = 8 × 10⁹ M^?1 s^?1) and electrons with a lower rate constant (k_e‐(aq) = 3.0 × 10⁸ M^?1 s^?1), doubled the crystal dose tolerance, whereas 1,4‐benzoquinone, which also scavenges both OH radicals (k_OH = 1.2 × 10⁹ M^?1 s^?1) and electrons (k_e‐(aq) = 1.2 × 10¹⁰ M^?1 s^?1), offered a ninefold increase in dose tolerance at the dose rates used. Pivotally, these preliminary results on a limited number of samples show that the two scavengers also induced a striking change in the dose dependence of the intensity decay from a first‐order to a zeroth‐order process.     

The 3D characteristics of post‐traumatic syringomyelia in a rat model: a propagation‐based synchrotron radiation microtomography study

Many published literature sources have described the histopathological characteristics of post‐traumatic syringomyelia (PTS). However, three‐dimensional (3D) visualization studies of PTS have been limited due to the lack of reliable 3D imaging techniques. In this study, the imaging efficiency of propagation‐based synchrotron radiation microtomography (PB‐SRµCT) was determined to detect the 3D morphology of the cavity and surrounding microvasculature network in a rat model of PTS. The rat model of PTS was established using the infinite horizon impactor to produce spinal cord injury (SCI), followed by a subarachnoid injection of kaolin to produce arachnoiditis. PB‐SRµCT imaging and histological examination, as well as fluorescence staining, were conducted on the animals at the tenth week after SCI. The 3D morphology of the cystic cavity was vividly visualized using PB‐SRµCT imaging. The quantitative parameters analyzed by PB‐SRµCT, including the lesion and spared spinal cord tissue area, the minimum and maximum diameters in the cystic cavity, and cavity volume, were largely consistent with the results of the histological assessment. Moreover, the 3D morphology of the cavity and surrounding angioarchitecture could be simultaneously detected on the PB‐SRµCT images. This study demonstrated that high‐resolution PB‐SRµCT could be used for the 3D visualization of trauma‐induced spinal cord cavities and provides valuable quantitative data for cavity characterization. PB‐SRµCT could be used as a reliable imaging technique and offers a novel platform for tracking cavity formation and morphological changes in an experimental animal model of PTS.