A mail-in data collection system at SPring-8, which is a web application with automated beamline operation, has been developed.
A mail-in data collection system makes it possible for beamline users to collect diffraction data without visiting a synchrotron facility. In the mail-in data collection system at SPring-8, users pack crystals into sample trays and send the trays to SPring-8 via a courier service as the first step. Next, the user specifies measurement conditions and checks the diffraction images via the Internet. The user can also collect diffraction data using an automated sample changer robot and beamline control software. For distant users there is a newly developed data management system, D-Cha. D-Cha provides a graphical user interface that enables the user to specify the experimental conditions for samples and to check and download the diffraction images using a web browser. This system is now in routine operation and is contributing to high-throughput beamline operation.
mail-in data collection; high-throughput data collection; beamline automation; web application; database system
The BL-17A macromolecular crystallography beamline at the Photon Factory was updated to improve the accuracy of diffraction experiments conducted using tiny crystals.
BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data. The stability of the beam intensities at the sample position was recently improved by modifying the monochromator. The diffractometer has also been improved. A new detector table was installed to prevent distortions in the diffractometer’s base during the repositioning of the diffractometer detector. A new pinhole system and an on-axis viewing system were installed to improve the X-ray beam profile at the sample position and the centering of tiny crystal samples.
macromolecular crystallography; beamline
MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides.
The design and features of a beamline control software system for macromolecular crystallography (MX) experiments developed at the European Synchrotron Radiation Facility (ESRF) are described. This system, MxCuBE, allows users to easily and simply interact with beamline hardware components and provides automated routines for common tasks in the operation of a synchrotron beamline dedicated to experiments in MX. Additional functionality is provided through intuitive interfaces that enable the assessment of the diffraction characteristics of samples, experiment planning, automatic data collection and the on-line collection and analysis of X-ray emission spectra. The software can be run in a tandem client-server mode that allows for remote control and relevant experimental parameters and results are automatically logged in a relational database, ISPyB. MxCuBE is modular, flexible and extensible and is currently deployed on eight macromolecular crystallography beamlines at the ESRF. Additionally, the software is installed at MAX-lab beamline I911-3 and at BESSY beamline BL14.1.
automation; macromolecular crystallography; synchrotron beamline control; graphical user interface
Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented.
The measurement of X-ray diffraction data from macromolecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals.
macromolecular crystallography; microcrystallography; X-ray beamlines; synchrotron radiation
A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services.
The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal reorientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.
workflows; automation; data processing; macromolecular crystallography; experimental protocols; characterization; reorientation; radiation damage
A system for the automatic reduction of single- and multi-position macromolecular crystallography data is presented.
The development of automated high-intensity macromolecular crystallography (MX) beamlines at synchrotron facilities has resulted in a remarkable increase in sample throughput. Developments in X-ray detector technology now mean that complete X-ray diffraction datasets can be collected in less than one minute. Such high-speed collection, and the volumes of data that it produces, often make it difficult for even the most experienced users to cope with the deluge. However, the careful reduction of data during experimental sessions is often necessary for the success of a particular project or as an aid in decision making for subsequent experiments. Automated data reduction pipelines provide a fast and reliable alternative to user-initiated processing at the beamline. In order to provide such a pipeline for the MX user community of the European Synchrotron Radiation Facility (ESRF), a system for the rapid automatic processing of MX diffraction data from single and multiple positions on a single or multiple crystals has been developed. Standard integration and data analysis programs have been incorporated into the ESRF data collection, storage and computing environment, with the final results stored and displayed in an intuitive manner in the ISPyB (information system for protein crystallography beamlines) database, from which they are also available for download. In some cases, experimental phase information can be automatically determined from the processed data. Here, the system is described in detail.
automation; data processing; macromolecular crystallography; computer programs
Optical trapping has successfully been applied to select and mount microcrystals for subsequent X-ray diffraction experiments.
X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 µm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.
laser tweezers; optical trapping; microcrystals; crystal manipulation; sample holders
A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material.
The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.
membrane proteins; lipid cubic phase; microradiography; microtomography
The collection of absorption and Raman spectroscopic data correlated with X-ray diffraction data allows investigators to understand the atomic structure as well as the electronic and vibrational characteristics of their samples, to identify transiently formed intermediates and to explore mechanistic questions. Raman spectroscopy instrumentation at beamline X26-C at the NSLS is currently available to the general user population.
Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data.
Raman; single-crystal spectroscopy; X-ray diffraction
The instrumentation and methods available for collecting almost simultaneous single-crystal electronic absorption correlated with X-ray diffraction data at NSLS beamline X26-C are reviewed, as well as a very brief outline of its Raman spectroscopy capability.
The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.
metalloenzymes; cofactors; electronic absorption spectroscopy; Raman spectroscopy
At the Photon Factory macromolecular crystallography beamlines, two new functions, remote monitoring and diffraction image evaluation, have been developed and installed on the beamline controlling system STARS (simple transmission and retrieval system).
Owing to recent advances in high-throughput technology in macromolecular crystallography beamlines, such as high-brilliant X-ray sources, high-speed readout detectors and robotics, the number of samples that can be examined in a single visit to the beamline has increased dramatically. In order to make these experiments more efficient, two functions, remote monitoring and diffraction image evaluation, have been implemented in the macromolecular crystallography beamlines at the Photon Factory (PF). Remote monitoring allows scientists to participate in the experiment by watching from their laboratories, without having to come to the beamline. Diffraction image evaluation makes experiments easier, especially when using the sample exchange robot. To implement these two functions, two independent clients have been developed that work specifically for remote monitoring and diffraction image evaluation. In the macromolecular crystallography beamlines at PF, beamline control is performed using STARS (simple transmission and retrieval system). The system adopts a client–server style in which client programs communicate with each other through a server process using the STARS protocol. This is an advantage of the extension of the system; implementation of these new functions required few modifications of the existing system.
macromolecular crystallography; beamline control system; remote monitoring; diffraction image evaluation
The Hard X-ray Nanoprobe Beamline is a precision platform for scanning probe and full-field microscopy with 3–30 keV X-rays. A combination of high-stability X-ray optics and precision motion sensing and control enables detailed studies of the internal features of samples with resolutions approaching 30 nm.
The Hard X-ray Nanoprobe Beamline (or Nanoprobe Beamline) is an X-ray microscopy facility incorporating diffraction, fluorescence and full-field imaging capabilities designed and operated by the Center for Nanoscale Materials and the Advanced Photon Source at Sector 26 of the Advanced Photon Source at Argonne National Laboratory. This facility was constructed to probe the nanoscale structure of biological, environmental and material sciences samples. The beamline provides intense focused X-rays to the Hard X-ray Nanoprobe (or Nanoprobe) which incorporates Fresnel zone plate optics and a precision laser sensing and control system. The beamline operates over X-ray energies from 3 to 30 keV, enabling studies of most elements in the periodic table, with a particular emphasis on imaging transition metals.
X-ray nanoprobe; X-ray microscopy; zone plate; X-ray fluorescence; nanodiffraction; nanotomography
Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented.
Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.
kappa goniometer; crystal alignment; data-collection strategies
The ultimate goal of synchrotron data collection is to obtain the best possible data from the best available crystals, and the combination of automation and remote access at Stanford Synchrotron Radiation Lightsource (SSRL) has revolutionized the way in which scientists achieve this goal. This has also seen a change in the way novice crystallographers are trained in the use of the beamlines, and a wide range of remote tools and hands-on workshops are now offered by SSRL to facilitate the education of the next generation of protein crystallographers.
For the past five years, the Structural Molecular Biology group at the Stanford Synchrotron Radiation Lightsource (SSRL) has provided general users of the facility with fully remote access to the macromolecular crystallography beamlines. This was made possible by implementing fully automated beamlines with a flexible control system and an intuitive user interface, and by the development of the robust and efficient Stanford automated mounting robotic sample-changing system. The ability to control a synchrotron beamline remotely from the comfort of the home laboratory has set a new paradigm for the collection of high-quality X-ray diffraction data and has fostered new collaborative research, whereby a number of remote users from different institutions can be connected at the same time to the SSRL beamlines. The use of remote access has revolutionized the way in which scientists interact with synchrotron beamlines and collect diffraction data, and has also triggered a shift in the way crystallography students are introduced to synchrotron data collection and trained in the best methods for collecting high-quality data. SSRL provides expert crystallographic and engineering staff, state-of-the-art crystallography beamlines, and a number of accessible tools to facilitate data collection and in-house remote training, and encourages the use of these facilities for education, training, outreach and collaborative research.
protein crystallography; high-throughput screening; robotics; remote access; crystallographic education and training; outreach
A high-precision diffractometer with a synchrotron radiation microfocusing technique has been developed to investigate the crystal structure of a submicrometre-scale single grain of powder sample. The structure of a BaTiO3 single powder grain, of dimensions ∼600 × 600 × 300 nm, was determined.
A high-precision diffractometer has been developed for the structure analysis of a submicrometre-scale single grain of a powder sample at the SPring-8 BL40XU undulator beamline. The key design concept is the combination of a stable focused synchrotron radiation beam and the precise axis control of the diffractometer, which allows accurate diffraction intensity data of a submicrometre-scale single powder grain to be measured. The phase zone plate was designed to create a high-flux focused synchrotron radiation beam. A low-eccentric goniometer and high-precision sample positioning stages were adopted to ensure the alignment of a micrometre-scale focused synchrotron radiation beam onto the submicrometre-scale single powder grain. In order to verify the diffractometer performance, the diffraction pattern data of several powder grains of BaTiO3, of dimensions ∼600 × 600 × 300 nm, were measured. By identifying the diffraction data set of one single powder grain, the crystal structure was successfully determined with a reliable factor of 5.24%.
submicrometre X-ray beam; phase zone plate; X-ray diffraction; single-crystal structure analysis; powder diffraction
The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening and data collection. At the Stanford Synchrotron Radiation Laboratory (SSRL), a National User Facility which provides extremely brilliant X-ray photon beams for use in materials science, environmental science and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Automated Mounter, or SAM) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter’s home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.
protein crystallography; cryocrystallography; high-throughput screening; robotics; remote access
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.
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 × 25 µm focus. The resulting beam had sufficient flux for crystallographic data collection from samples smaller than 10 × 10 × 10 µm. Sample data were collected representing three different scenarios: (i) a complete 2.0 Å data set from a single strongly diffracting microcrystal, (ii) a complete and redundant 1.94 Å data set obtained by merging data from six microcrystals and (iii) a complete 2.24 Å data set from a needle-shaped crystal with less than 12 × 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 × 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.
mini-beam; microbeam; microcrystals; microdiffraction; high mosaicity; inhomogeneous crystal; signal-to-noise; crystal segment; beam divergence; streaky spots
An instrumentation and data analysis review of coherent diffraction microscopy at SPring-8 is given. This work will be of interest to those who want to apply coherent diffraction imaging to studies of materials science and biological samples.
Since the first demonstration of coherent diffraction microscopy in 1999, this lensless imaging technique has been experimentally refined by continued developments. Here, instrumentation and experimental procedures for measuring oversampled diffraction patterns from non-crystalline specimens using an undulator beamline (BL29XUL) at SPring-8 are presented. In addition, detailed post-experimental data analysis is provided that yields high-quality image reconstructions. As the acquisition of high-quality diffraction patterns is at least as important as the phase-retrieval procedure to guarantee successful image reconstructions, this work will be of interest for those who want to apply this imaging technique to materials science and biological samples.
coherent diffraction microscopy; coherent diffraction imaging; lensless imaging; oversampling; phase retrieval
Recent developments in X-ray crystallographic hardware related to structural biology research are presented and discussed.
While the majority of macromolecular X-ray data are currently collected using highly efficient beamlines at an ever-increasing number of synchrotrons, there is still a need for high-performance reliable systems for in-house experiments. In addition to crystal screening and optimization of data-collection parameters before a synchrotron trip, the home system allows the collection of data as soon as the crystals are produced to obtain the solution of novel structures, especially by the molecular-replacement method, and is invaluable in achieving the quick turnover that is often required for ligand-binding studies in the pharmaceutical industry. There has been a continuous evolution of X-ray sources, detectors and software developed for in-house use in recent years and a diverse range of tools for structural biology laboratories are available. An overview of the main directions of these developments and examples of specific solutions available to the macromolecular crystallography community are presented in this paper, showing that data collection ‘at home’ is still an attractive proposition complementing the use of synchrotron beamlines.
macromolecular crystallography; X-ray hardware; data collection
Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
autoPROC; data processing
High-quality, ice-free X-ray diffraction data were continuously collected while cryoprotectant-free thaumatin crystals were cooled at 0.1 K s−1 from 300 to 100 K. This establishes the feasibility of fully temperature controlled studies of protein structure and dynamics, and provides insight into how cooling creates crystal disorder.
Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s−1 – 103–104 times slower than in conventional flash cooling – while continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2°. Most of the mosaicity increase occurs above ∼200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3–6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.
protein crystallography; cryocrystallography; X-ray diffraction; slow cooling; temperature control
The Protein Crystallography Station user facility at Los Alamos National Laboratory not only offers open access to a high-performance neutron beamline, but also actively supports and develops new methods in protein expression, deuteration, purification, robotic crystallization and the synthesis of substrates with stable isotopes and provides assistance with data-reduction and structure-refinement software and comprehensive neutron structure analysis.
The Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. Neutron diffraction is a powerful technique for locating H atoms and can therefore provide unique information about how biological macromolecules function and interact with each other and smaller molecules. Users of the PCS have access to neutron beam time, deuteration facilities, the expression of proteins and the synthesis of substrates with stable isotopes and also support for data reduction and structure analysis. The beamline exploits the pulsed nature of spallation neutrons and a large electronic detector in order to collect wavelength-resolved Laue patterns using all available neutrons in the white beam. The PCS user facility is described and highlights from the user program are presented.
Protein Crystallography Station; neutron macromolecular crystallography; spallation neutron sources; deuteration; user support
RapiData provides two days of high-level lectures, then two more of experimental work on several beamlines of the National Synchrotron Light Source, for about 50 students. This article provides details about the organization of the course and tells some of the reasons for its success.
RapiData provides two days of high-level lectures, then two more of experimental work on several beamlines of the National Synchrotron Light Source, for about 50 students. Students are invited to bring their own research projects for measurement, and about half of them do. The students frequently solve half a dozen structures during the course. Tutorials by the lecturers run throughout the data-collection period. The crystal-preparation laboratory is popular for tutorials and practice, and often there is a beamline available for practice. This article provides details about the organization of the course and tells some of the reasons for its success.
crystallographic education; RapiData; macromolecular crystallography; NSLS
Lens- and fiber-coupled X-ray detectors with identical CCD chips were compared in their performance in high-resolution computed tomography experiments.
X-ray imaging detectors with an identical phosphor and a CCD chip but employing lens- and fiber-coupling between them have been compared. These are designed for X-ray imaging experiments, especially computed tomography, at the medium-length beamline at the SPring-8 synchrotron radiation facility. It was found that the transmittance of light to the CCD is about four times higher in the fiber-coupled detector. The uniformity of response in the lens-coupled detector has a global shading of up to 40%, while pixel-to-pixel variation owing to a chicken-wire pattern was dominant in the fiber-coupled detector. Apart from the higher transmittance, the fiber-coupled detector has a few characteristics that require attention when it is used for computed tomography, which are browning of the fiber, discontinuity in the image, image distortion, and dark spots in the chicken-wire pattern. Thus, it is most suitable for high-speed tomography of samples that tend to deform, for example biological and soft materials.
X-ray imaging; fiber-coupled CCD; lens-coupled CCD; microtomography
AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken.
Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P65, with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement.
N-acetyltransferases; AAC(6′)-Im; aminoglycoside resistance