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1.  Mail-in data collection at SPring-8 protein crystallography beamlines 
Journal of Synchrotron Radiation  2008;15(Pt 3):288-291.
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.
PMCID: PMC2394786  PMID: 18421161
mail-in data collection; high-throughput data collection; beamline automation; web application; database system
2.  Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization 
Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A−1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.
PMCID: PMC3832202  PMID: 24260038
3.  Matching X-ray beam and detector properties to protein crystals of different perfection 
Journal of Synchrotron Radiation  2014;21(Pt 3):537-546.
Expressions are given to match X-ray data collection facilities to the intrinsic diffraction properties of crystals with different degrees of perfection.
An analysis is given of the effect of different beam and detector parameters on the sharpness of recorded diffraction features for macromolecular crystals of different quality. The crystal quality parameters include crystal strain, crystal or mosaic block size and mosaic block misorientation. Calculations are given for instrument parameters such as angular resolution of the detector, beam divergence and wavelength bandpass to be matched to the intrinsic diffraction properties from these crystals with the aim of obtaining the best possible data out of each crystal. Examples are given using typical crystal imperfections obtained from the literature for both room-temperature and cryo-cooled crystals. Possible implications for the choice of X-ray source, beamline design, detector specifications, instrument set-up and data processing are discussed, together with the limitations of the approach.
PMCID: PMC3998814  PMID: 24763643
crystal perfection; synchrotron beam properties; detectors
4.  Improvements toward highly accurate diffraction experiments at the macromolecular micro-crystallography beamline BL-17A 
Journal of Synchrotron Radiation  2013;20(Pt 6):938-942.
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.
PMCID: PMC3795560  PMID: 24121344
macromolecular crystallography; beamline
5.  MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments 
Journal of Synchrotron Radiation  2010;17(Pt 5):700-707.
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.
PMCID: PMC3025540  PMID: 20724792
automation; macromolecular crystallography; synchrotron beamline control; graphical user interface
6.  The design of macromolecular crystallography diffraction experiments 
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.
PMCID: PMC3069741  PMID: 21460444
macromolecular crystallography; microcrystallography; X-ray beamlines; synchrotron radiation
7.  The use of workflows in the design and implementation of complex experiments in macromolecular crystallography 
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.
PMCID: PMC3413211  PMID: 22868763
workflows; automation; data processing; macromolecular crystallography; experimental protocols; characterization; reorientation; radiation damage
8.  AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery 
New software has been developed for automating the experimental and data-processing stages of fragment-based drug discovery at a macromolecular crystallography beamline. A new workflow-automation framework orchestrates beamline-control and data-analysis software while organizing results from multiple samples.
AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallo­graphy steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference Fourier maps.
PMCID: PMC3640469  PMID: 23633588
AutoDrug; fragment-based drug discovery; workflow automation
9.  Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF 
Journal of Applied Crystallography  2013;46(Pt 3):804-810.
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.
PMCID: PMC3654316  PMID: 23682196
automation; data processing; macromolecular crystallography; computer programs
10.  Microcrystal manipulation with laser tweezers 
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.
PMCID: PMC3689533  PMID: 23793156
laser tweezers; optical trapping; microcrystals; crystal manipulation; sample holders
11.  Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging 
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.
PMCID: PMC3689528  PMID: 23793151
membrane proteins; lipid cubic phase; microradiography; microtomography
12.  Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS 
Journal of Synchrotron Radiation  2010;18(Pt 1):37-40.
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.
PMCID: PMC3004251  PMID: 21169688
Raman; single-crystal spectroscopy; X-ray diffraction
13.  Correlated single-crystal electronic absorption spectroscopy and X-ray crystallography at NSLS beamline X26-C 
Journal of Synchrotron Radiation  2011;18(Pt 3):358-366.
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.
PMCID: PMC3083912  PMID: 21525643
metalloenzymes; cofactors; electronic absorption spectroscopy; Raman spectroscopy
14.  Implementation of remote monitoring and diffraction evaluation systems at the Photon Factory macromolecular crystallography beamlines 
Journal of Synchrotron Radiation  2008;15(Pt 3):296-299.
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.
PMCID: PMC2394808  PMID: 18421163
macromolecular crystallography; beamline control system; remote monitoring; diffraction image evaluation
15.  A hard X-ray nanoprobe beamline for nanoscale microscopy 
Journal of Synchrotron Radiation  2012;19(Pt 6):1056-1060.
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.
PMCID: PMC3579591  PMID: 23093770
X-ray nanoprobe; X-ray microscopy; zone plate; X-ray fluorescence; nanodiffraction; nanotomography
16.  Dynamic full-field infrared imaging with multiple synchrotron beams 
Analytical chemistry  2013;85(7):3599-3605.
Microspectroscopic imaging in the infrared (IR) spectral region allows for the examination of spatially resolved chemical composition on the microscale. More than a decade ago, it was demonstrated that diffraction limited spatial resolution can be achieved when an apertured, single pixel IR microscope is coupled to the high brightness of a synchrotron light source. Nowadays, many IR microscopes are equipped with multi-pixel Focal Plane Array (FPA) detectors, which dramatically improve data acquisition times for imaging large areas. Recently, progress been made toward efficiently coupling synchrotron IR beamlines to multi-pixel detectors, but they utilize expensive and highly customized optical schemes. Here we demonstrate the development and application of a simple optical configuration that can be implemented on most existing synchrotron IR beamlines in order to achieve full-field IR imaging with diffraction-limited spatial resolution. Specifically, the synchrotron radiation fan is extracted from the bending magnet and split into four beams that are combined on the sample, allowing it to fill a large section of the FPA. With this optical configuration, we are able to oversample an image by more than a factor of two, even at the shortest wavelengths, making image restoration through deconvolution algorithms possible. High chemical sensitivity, rapid acquisition times, and superior signal-to-noise characteristics of the instrument are demonstrated. The unique characteristics of this setup enabled the real time study of heterogeneous chemical dynamics with diffraction-limited spatial resolution for the first time.
PMCID: PMC3652324  PMID: 23458231
synchrotron; infrared microspectroscopy; imaging; FTIR; focal plane array; dynamics
17.  The use of a mini-κ goniometer head in macromolecular crystallography diffraction experiments 
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.
PMCID: PMC3689527  PMID: 23793150
kappa goniometer; crystal alignment; data-collection strategies
18.  Remote access to crystallography beamlines at SSRL: novel tools for training, education and collaboration 
Journal of Applied Crystallography  2010;43(Pt 5):1261-1270.
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.
PMCID: PMC3238386  PMID: 22184477
protein crystallography; high-throughput screening; robotics; remote access; crystallographic education and training; outreach
19.  X-ray diffractometry for the structure determination of a submicrometre single powder grain 
Journal of Synchrotron Radiation  2009;16(Pt 3):352-357.
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%.
PMCID: PMC2678016  PMID: 19395798
submicrometre X-ray beam; phase zone plate; X-ray diffraction; single-crystal structure analysis; powder diffraction
20.  The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL 
JALA (Charlottesville, Va.)  2008;13(6):335-343.
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.
PMCID: PMC2654326  PMID: 19956359
protein crystallography; cryocrystallography; high-throughput screening; robotics; remote access
21.  A 7 µm mini-beam improves diffraction data from small or imperfect crystals of macromolecules 
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.
PMCID: PMC2631116  PMID: 18391409
mini-beam; microbeam; microcrystals; microdiffraction; high mosaicity; inhomogeneous crystal; signal-to-noise; crystal segment; beam divergence; streaky spots
22.  Coherent diffraction microscopy at SPring-8: instrumentation, data acquisition and data analysis 
Journal of Synchrotron Radiation  2011;18(Pt 2):293-298.
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.
PMCID: PMC3042331  PMID: 21335919
coherent diffraction microscopy; coherent diffraction imaging; lensless imaging; oversampling; phase retrieval
23.  Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1 
Human Cockayne syndrome protein A has been cocrystallized with human DNA damage-binding protein 1 and data have been collected to 2.9 Å resolution.
Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility.
PMCID: PMC3253832  PMID: 22232169
Cockayne syndrome protein A; DNA damage-binding protein 1
24.  Collecting data in the home laboratory: evolution of X-ray sources, detectors and working practices 
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.
PMCID: PMC3689531  PMID: 23793154
macromolecular crystallography; X-ray hardware; data collection
25.  Data processing and analysis with the autoPROC toolbox 
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.
PMCID: PMC3069744  PMID: 21460447
autoPROC; data processing

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