Energy optimization of a regular macromolecular crystallography beamline for ultra‐high‐resolution crystallography

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19‐ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower‐ and higher‐energy harmonic contamination. A Pd‐coated mirror and Al attenuators acted as effective low‐ and high‐bandpass filters. The resulting flux at 30 keV, although significantly lower than with X‐ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.     

Automatic crystal centring procedure at the SSRF macromolecular crystallography beamline

X‐ray diffraction is a common technique for determining crystal structures. The average time needed for the solution of a protein structure has been drastically reduced by a number of recent experimental and theoretical developments. Since high‐throughput protein crystallography benefits from full automation of all steps that are carried out on a synchrotron beamline, an automatic crystal centring procedure is important for crystallographic beamlines. Fully automatic crystal alignment involves the application of optical methods to identify the crystal and move it onto the rotation axis and into the X‐ray beam. Crystal recognition has complex dependencies on the illumination, crystal size and viewing angles due to effects such as local shading, inter‐reflections and the presence of antifreezing elements. Here, a rapid procedure for crystal centring with multiple cameras using region segment thresholding is reported. Firstly, a simple illumination‐invariant loop recognition and classification model is used by slicing a low‐magnification loop image into small region segments, then classifying the loop into different types and aligning it to the beam position using feature vectors of the region segments. Secondly, an edge detection algorithm is used to find the crystal sample in a high‐magnification image using region segment thresholding. Results show that this crystal centring method is extremely successful under fluctuating light states as well as for poorly frozen and opaque samples. Moreover, this crystal centring procedure is successfully integrated into the enhanced Blu‐Ice data collection system at beamline BL17U1 at the Shanghai Synchrotron Radiation Facility as a routine method for an automatic crystal screening procedure.     

A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results

Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are 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.     

Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography

The sulfur SAD phasing method allows the determination of protein structures de novo without reference to derivatives such as Se‐methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimeric Achromobacter cycloclastes nitrite reductase (AcNiR), which contains a near average proportion of sulfur‐containing residues and two Cu atoms per subunit. Experiments using X‐ray wavelengths in the range 1.9–2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful for automated structure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X‐ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long‐wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ～1.25% at ～2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ～0.75 Å) for AcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.     

A new dimension in structural biology: fully fledged high-pressure macromolecular crystallography

The amount of accurate crystallographic data currently available on macromolecular structures at high pressure is extremely limited, mainly due to the lack of appropriate instrumentation and X-ray sources. A technical breakthrough has been achieved with a set-up at the ESRF ID30 beamline equipped with a diamond anvil cell and a large imaging plate, and taking advantage from undulators providing a quasi-plane wave of ultrashort wavelength X-rays. The accessible pressure range is increased by nearly one order of magnitude with respect to beryllium cells. A nearly perfect long-range order is preserved in protein and virus crystals compressed below the denaturation pressure. The quality of diffraction data collected at high pressure can meet usual standards, especially in the case of high symmetry space groups.

An overview of main results and ongoing studies is given, including the 7?kbar structure of hen egg-white lysozyme refined at 1.6?Å resolution and structural investigations of cowpea mosaic virus (CPMV), the first macromolecular assembly investigated at high pressure by single crystal X-ray diffraction. The ordering effect of high pressure on the molecular packing of disordered P23 crystals was highlighted. The 2.8?Å resolution structure of CPMV at 3.3?kbar was fully refined using high completeness data collected on pressure-ordered I23 crystals.     

Are you centered? An automatic crystal‐centering method for high‐throughput macromolecular crystallography

Crystal centering is a key step in macromolecular X‐ray crystallography experiments. A new method using image‐processing and machine‐vision techniques allows the centering of small crystals in the X‐ray beam. This method positions crystals even when the loop is initially out of the camera's field of view and adapts to the difficulty of the experiment. The process has been tested on many diverse crystals with a 93% success rate when compared with manual centering.     

A 3 × 6 arrayed CCD X‐ray detector for continuous rotation method in macromolecular crystallography

A 3 × 6 arrayed charge‐coupled device (CCD) X‐ray detector has been developed for the continuous‐rotation method in macromolecular crystallography at the Photon Factory. The detector has an area of 235.9 mm × 235.9 mm and a readout time of 1.9 s. The detector is made of a 3 × 6 array of identical modules, each module consisting of a fiber‐optic taper (FOT), a CCD sensor and a readout circuit. The outputs from 18 CCDs are read out in parallel and are then digitized by 16‐bit analog‐to‐digital converters. The advantage of this detector over conventional FOT‐coupled CCD detectors is the unique CCD readout scheme (frame transfer) which enables successive X‐ray exposures to be recorded without interruption of the sample crystal rotation. A full data set of a lysozyme crystal was continuously collected within 360 s (180° rotation, 3 s/1.5° frame). The duty‐cycle ratio of the X‐ray exposure to the data collection time was almost 100%. The combination of this detector and synchrotron radiation is well suited to rapid and continuous data collection in macromolecular crystallography.     

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.     

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.     

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.     

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.     

The impact of synchrotron radiation in protein crystallography

The Pohang Light Source (PLS) at the Pohang Accelerator Laboratory (PAL) is a third-generation light source, the only synchrotron radiation facility in Korea, and the fifth machine of its kind in the world (see Figure 1). In 1988, PAL was organized for the construction of the PLS. Ground-breaking was celebrated in 1991, and PLS construction was completed in 1994. In 1995, the PLS opened two beamlines to public users. The PLS was initially operated at 2.0 GeV in 1995. Since 2002, the energy of the electron beam has been upgraded to 2.5 GeV (see Table 1 for the principal parameters of PLS). Remarkable increases in the number of beamlines, users, and scientific results have been achieved since the opening of the PLS in 1995. Two or three beamlines have been added each year for the past 15 years, and as of February 2009 we have in total 27 beamlines in operation and 3 beamlines under construction, which will be completed by the end of 2009 (Figures 2 and 3).     

The RATIO method for time‐resolved Laue crystallography

A RATIO method for analysis of intensity changes in time‐resolved pump–probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam.     

Slotted carbide anvils: improved X-ray access for synchrotron-based multi-anvil experiments

We present a new technique to allow enhanced X-ray access to synchrotron-based multi-anvil experiments with tungsten carbide anvils. Slots of 2 mm width and 1 mm depth are cut into the four anvils which are in the beam path along their ? 110? diagonals and these slots are filled with baked pyrophyllite to produce a region in the anvils which is X-ray transparent. We have tested this arrangement using a standard 10/5 cell assembly and the X-ray window remains open, allowing imaging of a 2.5 by 2 mm section of the cell to at least 20 GPa. There is no evidence that the slots reduce the life of the anvils and the recovered slotted anvils have been used in subsequent experiments.     

Review on applications of synchrotron-based X-ray techniques in materials characterization

Synchrotron radiation (SR), as a result of its high-intensity, brilliant, monochromatic, and collimated beams, is becoming one of the most crucial components of research in various fields of materials science such as nanomaterials, biomaterials, and energy materials. SR-based characterization methods can be employed to analyze different systems such as powders, thin films, and bulk forms having complex crystalline or amorphous structures. In this review, peculiarities of SR are briefly explained. Moreover, various techniques carried out utilizing this instrument for material characterization such as X-ray powder diffraction, grazing-incidence X-ray diffraction, small/wide-angle X-ray scattering, X-ray absorption spectroscopy, different techniques of X-ray imaging, X-ray photoelectron spectroscopy, and X-ray microprobes/nanoprobes are presented. As a result, by shedding light on the advantages of SR and its superiority to the equivalent laboratory experiments, researchers are recommended to exploit the capabilities of this invaluable tool in their materials characterization.