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
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
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
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
Statistical analysis system (SAS) is the most comprehensive statistical analysis software package in the world. It offers data analysis for almost all experiments under various statistical models. Each analysis is performed using a particular subroutine, called a procedure (PROC). For example, PROC ANOVA performs analysis of variances. PROC QTL is a user-defined SAS procedure for mapping quantitative trait loci (QTL). It allows users to perform QTL mapping for continuous and discrete traits within the SAS platform. Users of PROC QTL are able to take advantage of all existing features offered by the general SAS software, for example, data management and graphical treatment. The current version of PROC QTL can perform QTL mapping for all line crossing experiments using maximum likelihood (ML), least square (LS), iteratively reweighted least square (IRLS), Fisher scoring (FISHER), Bayesian (BAYES), and empirical Bayes (EBAYES) methods.
A repetitive measurement of the same diffraction image allows to judge the performance of a data collection facility.
The accuracy of X-ray diffraction data depends on the properties of the crystalline sample and on the performance of the data-collection facility (synchrotron beamline elements, goniostat, detector etc.). However, it is difficult to evaluate the level of performance of the experimental setup from the quality of data sets collected in rotation mode, as various crystal properties such as mosaicity, non-uniformity and radiation damage affect the measured intensities. A multiple-image experiment, in which several analogous diffraction frames are recorded consecutively at the same crystal orientation, allows minimization of the influence of the sample properties. A series of 100 diffraction images of a thaumatin crystal were measured on the SBC beamline 19BM at the APS (Argonne National Laboratory). The obtained data were analyzed in the context of the performance of the data-collection facility. An objective way to estimate the uncertainties of individual reflections was achieved by analyzing the behavior of reflection intensities in the series of analogous diffraction images. The multiple-image experiment is found to be a simple and adequate method to decompose the random errors from the systematic errors in the data, which helps in judging the performance of a data-collection facility. In particular, displaying the intensity as a function of the frame number allows evaluation of the stability of the beam, the beamline elements and the detector with minimal influence of the crystal properties. Such an experiment permits evaluation of the highest possible data quality potentially achievable at the particular beamline.
diffraction data precision; signal-to-noise ratio; measurement uncertainty; beamline performance
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
The DICHROWEB web server enables on-line analyses of circular dichroism (CD) spectroscopic data, providing calculated secondary structure content and graphical analyses comparing calculated structures and experimental data. The server is located at http://www.cryst.bbk.ac.uk/cdweb and may be accessed via a password-limited user ID, available upon completion of a registration form. The server facilitates analyses using five popular algorithms and (currently) seven different reference databases by accepting data in a user-friendly manner in a wide range of formats, including those output by both commercial CD instruments and synchrotron radiation-based circular dichroism beamlines, as well as those produced by spectral processing software packages. It produces as output calculated secondary structures, a goodness-of-fit parameter for the analyses, and tabular and graphical displays of experimental, calculated and difference spectra. The web pages associated with the server provide information on CD spectroscopic methods and terms, literature references and aids for interpreting the analysis results.
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
Gene expression microarrays are a prominent experimental tool in functional genomics which has opened the opportunity for gaining global, systems-level understanding of transcriptional networks. Experiments that apply this technology typically generate overwhelming volumes of data, unprecedented in biological research. Therefore the task of mining meaningful biological knowledge out of the raw data is a major challenge in bioinformatics. Of special need are integrative packages that provide biologist users with advanced but yet easy to use, set of algorithms, together covering the whole range of steps in microarray data analysis.
Here we present the EXPANDER 2.0 (EXPression ANalyzer and DisplayER) software package. EXPANDER 2.0 is an integrative package for the analysis of gene expression data, designed as a 'one-stop shop' tool that implements various data analysis algorithms ranging from the initial steps of normalization and filtering, through clustering and biclustering, to high-level functional enrichment analysis that points to biological processes that are active in the examined conditions, and to promoter cis-regulatory elements analysis that elucidates transcription factors that control the observed transcriptional response. EXPANDER is available with pre-compiled functional Gene Ontology (GO) and promoter sequence-derived data files for yeast, worm, fly, rat, mouse and human, supporting high-level analysis applied to data obtained from these six organisms.
EXPANDER integrated capabilities and its built-in support of multiple organisms make it a very powerful tool for analysis of microarray data. The package is freely available for academic users at
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
This paper describes FieldTrip, an open source software package that we developed for the analysis of MEG, EEG, and other electrophysiological data. The software is implemented as a MATLAB toolbox and includes a complete set of consistent and user-friendly high-level functions that allow experimental neuroscientists to analyze experimental data. It includes algorithms for simple and advanced analysis, such as time-frequency analysis using multitapers, source reconstruction using dipoles, distributed sources and beamformers, connectivity analysis, and nonparametric statistical permutation tests at the channel and source level. The implementation as toolbox allows the user to perform elaborate and structured analyses of large data sets using the MATLAB command line and batch scripting. Furthermore, users and developers can easily extend the functionality and implement new algorithms. The modular design facilitates the reuse in other software packages.
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
EMEGS (electromagnetic encephalography software) is a MATLAB toolbox designed to provide novice as well as expert users in the field of neuroscience with a variety of functions to perform analysis of EEG and MEG data. The software consists of a set of graphical interfaces devoted to preprocessing, analysis, and visualization of electromagnetic data. Moreover, it can be extended using a plug-in interface. Here, an overview of the capabilities of the toolbox is provided, together with a simple tutorial for both a standard ERP analysis and a time-frequency analysis. Latest features and future directions of the software development are presented in the final section.
We describe a collection of standardized image processing protocols for electron microscopy single-particle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.
The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide one potential route for other SAXS beamlines in need of robust and efficient beamline control software.
Biological small-angle X-ray scattering (SAXS) provides powerful complementary data for macromolecular crystallography (MX) by defining shape, conformation and assembly in solution. Although SAXS is in principle the highest throughput technique for structural biology, data collection is limited in practice by current data collection software. Here the adaption of beamline control software, historically developed for MX beamlines, for the efficient operation and high-throughput data collection at synchrotron SAXS beamlines is reported. The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the biological SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide a potential route for other SAXS beamlines in need of robust and efficient beamline control software. As a great deal of effort and optimization has gone into crystallographic software, the adaption and extension of crystallographic software may prove to be a general strategy to provide advanced SAXS software for the synchrotron community. In this way effort can be put into optimizing features for SAXS rather than reproducing those that have already been successfully implemented for the crystallographic community.
SAXS; software; beamline; control system; Blu-Ice; DCS; SIBYLS; GUI
► We present a new functional transcranial Doppler ultrasongraphy toolbox for Matlab. ► The basic features include multi-file summaries and laterality index calculation. ► Advanced features include behavioural and multi-session summaries. ► The toolbox is freely available under the GNU GPL license.
We present a description of a new software package, ‘dopOSCCI’, which summarises data from experimental studies where functional transcranial Doppler ultrasonography (fTCD) is used to compare hemispheric rates of blood flow in order to assess lateralization of a cognitive process. The software provides a graphical user interface to summarise analogue and digital data collected using Multi-Dop Doppler Ultrasound devices (DWL Multidop T2: manufacturer, DWL Elektronische Systeme, Singen, Germany). The unique aspects of dopOSCCI allow multi-file processing, multi-event marker processing, behavioural and multi-session summaries, image file data visualization, and tab-delimited output files which includes split-half, single-trial summaries and data quality variables. The Matlab based software is available under the GNU GPL license and can be accessed online at https://databank.ora.ox.ac.uk/general/datasets/dopOSCCI, the Oxford University DataBank.
Functional transcranial Doppler ultrasonography; Imaging; Blood flow velocity; Methods; Matlab
The post-experiment processing of X-ray Diffraction Microscopy data is often time-consuming and difficult. This is mostly due to the fact that even if a preliminary result has been reconstructed, there is no definitive answer as to whether or not a better result with more consistently retrieved phases can still be obtained. We show here that the first step in data analysis, the assembly of two-dimensional diffraction patterns from a large set of raw diffraction data, is crucial to obtaining reconstructions of highest possible consistency. We have developed software that automates this process and results in consistently accurate diffraction patterns. We have furthermore derived some criteria of validity for a tool commonly used to assess the consistency of reconstructions, the phase retrieval transfer function, and suggest a modified version that has improved utility for judging reconstruction quality.
Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data.
Statistical bioinformatics is the study of biological data sets obtained by new micro-technologies by means of proper statistical methods. For a better understanding of environmental adaptations of proteins, orthologous sequences from different habitats may be explored and compared. The main goal of the DeltaProt Toolbox is to provide users with important functionality that is needed for comparative screening and studies of extremophile proteins and protein classes. Visualization of the data sets is also the focus of this article, since visualizations can play a key role in making the various relationships transparent. This application paper is intended to inform the reader of the existence, functionality, and applicability of the toolbox.
We present the DeltaProt Toolbox, a software toolbox that may be useful in importing, analyzing and visualizing data from multiple alignments of proteins. The toolbox has been written in MATLAB™ to provide an easy and user-friendly platform, including a graphical user interface, while ensuring good numerical performance. Problems in genome biology may be easily stated thanks to a compact input format. The toolbox also offers the possibility of utilizing structural information from the SABLE or other structure predictors. Different sequence plots can then be viewed and compared in order to find their similarities and differences. Detailed statistics are also calculated during the procedure.
The DeltaProt package is open source and freely available for academic, non-commercial use. The latest version of DeltaProt can be obtained from http://services.cbu.uib.no/software/deltaprot/. The website also contains documentation, and the toolbox comes with real data sets that are intended for training in applying the models to carry out bioinformatical and statistical analyses of protein sequences.
Equipped with the new algorithms proposed here, DeltaProt serves as an auxiliary analysis tool capable of visualizing and comparing orthologus protein sequences. The framework of the algorithms also enables easy incorporation of extra information on structure, if such data is available.
Two-dimensional data needs to be processed and analysed in almost any experimental laboratory. Some tasks in this context may be performed with generic software such as spreadsheet programs which are available ubiquitously, others may require more specialised software that requires paid licences. Additionally, more complex software packages typically require more time by the individual user to understand and operate. Practical and convenient graphical data analysis software in Java with a user-friendly interface are rare.
We have developed SDAR, a Java application to analyse two-dimensional data with an intuitive graphical user interface. A smart ASCII parser allows import of data into SDAR without particular format requirements. The centre piece of SDAR is the Java class GraphPanel which provides methods for generic tasks of data visualisation. Data can be manipulated and analysed with respect to the most common operations experienced in an experimental biochemical laboratory. Images of the data plots can be generated in SVG-, TIFF- or PNG-format. Data exported by SDAR is annotated with commands compatible with the Grace software.
Since SDAR is implemented in Java, it is truly cross-platform compatible. The software is easy to install, and very convenient to use judging by experience in our own laboratories. It is freely available to academic users at
http://www.structuralchemistry.org/pcsb/. To download SDAR, users will be asked for their name, institution and email address. A manual, as well as the source code of the GraphPanel class can also be downloaded from this site.
SaSAT (Sampling and Sensitivity Analysis Tools) is a user-friendly software package for applying uncertainty and sensitivity analyses to mathematical and computational models of arbitrary complexity and context. The toolbox is built in Matlab®, a numerical mathematical software package, and utilises algorithms contained in the Matlab® Statistics Toolbox. However, Matlab® is not required to use SaSAT as the software package is provided as an executable file with all the necessary supplementary files. The SaSAT package is also designed to work seamlessly with Microsoft Excel but no functionality is forfeited if that software is not available. A comprehensive suite of tools is provided to enable the following tasks to be easily performed: efficient and equitable sampling of parameter space by various methodologies; calculation of correlation coefficients; regression analysis; factor prioritisation; and graphical output of results, including response surfaces, tornado plots, and scatterplots. Use of SaSAT is exemplified by application to a simple epidemic model. To our knowledge, a number of the methods available in SaSAT for performing sensitivity analyses have not previously been used in epidemiological modelling and their usefulness in this context is demonstrated.
A systematic analysis of diffraction data of 11 different lysozyme crystals (used for cisplatin and carboplatin binding studies), obtained with four diffraction data processing software packages and from two different diffractometers, serves as a pilot study for archiving raw diffraction data and associated metadata. The availability of the raw diffraction images allows for independent assessment of software packages.
The International Union of Crystallography has for many years been advocating archiving of raw data to accompany structural papers. Recently, it initiated the formation of the Diffraction Data Deposition Working Group with the aim of developing standards for the representation of these data. A means of studying this issue is to submit exemplar publications with associated raw data and metadata. A recent study on the effects of dimethyl sulfoxide on the binding of cisplatin and carboplatin to histidine in 11 different lysozyme crystals from two diffractometers led to an investigation of the possible effects of the equipment and X-ray diffraction data processing software on the calculated occupancies and B factors of the bound Pt compounds. 35.3 Gb of data were transferred from Manchester to Utrecht to be processed with EVAL. A systematic comparison shows that the largest differences in the occupancies and B factors of the bound Pt compounds are due to the software, but the equipment also has a noticeable effect. A detailed description of and discussion on the availability of metadata is given. By making these raw diffraction data sets available via a local depository, it is possible for the diffraction community to make their own evaluation as they may wish.
data exchange; data archiving; metadata
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
A detailed understanding of chemical and biological function and the mechanisms underlying the activities ultimately requires atomic-resolution structural data. Diffraction-based techniques such as single-crystal X-ray crystallography, electron microscopy and neutron diffraction are well established and have paved the road to the stunning successes of modern-day structural biology. The major advances achieved in the last 20 years in all aspects of structural research, including sample preparation, crystallization, the construction of synchrotron and spallation sources, phasing approaches and high-speed computing and visualization, now provide specialists and non-specialists alike with a steady flow of molecular images of unprecedented detail. The present chapter combines a general overview of diffraction methods with a step-by-step description of the process of a single-crystal X-ray structure determination experiment, from chemical synthesis or expression to phasing and refinement, analysis and quality control. For novices it may serve as a stepping-stone to more in-depth treatises of the individual topics. Readers relying on structural information for interpreting functional data may find it a useful consumer guide.