A revised version of Table 2 of Nanao et al. [J. Synchrotron Rad. (2022). 29, 581-590] is provided.
ID23-2 is a fixed-energy (14.2â keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF-EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013â photonsâ s-1) focused microbeam (minimum size 1.5â µm × 3â µm full width at half-maximum). The sample environment now includes a FLEX-HCD sample changer/storage system, as well as a state-of-the-art MD3Up high-precision multi-axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments.
Lenses , Synchrotrons , Crystallography, X-Ray , Data Collection , Macromolecular Substances/chemistry
ID29 is an ESRF undulator beamline with a routinely accessible energy range of between 20.0â keV and 6.0â keV (λ = 0.62â Å to 2.07â Å) dedicated to the use of anomalous dispersion techniques in macromolecular crystallography. Since the beamline was first commissioned in 2001, ID29 has, in order to provide an improved service to both its academic and proprietary users, been the subject of almost continuous upgrade and refurbishment. It is now also the home to the ESRF Cryobench facility, ID29S. Here, the current status of the beamline is described and plans for its future are briefly outlined.
Macromolecular Substances/chemistry , Synchrotrons/instrumentation , Trypsin/chemistry , X-Ray Diffraction
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.
Crystallography, X-Ray/methods , Software , Synchrotrons , Carboxylic Ester Hydrolases/chemistry , Databases, Factual , Macromolecular Substances/chemistry , Spectrometry, X-Ray Emission , Thermolysin/chemistry
Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users.
Proteins/analysis , X-Ray Diffraction/instrumentation , X-Ray Diffraction/methods , Animals , Chickens , Crystallization , Escherichia coli/chemistry , Models, Molecular , Protein Structure, Tertiary , Proteins/chemistry , Water/chemistry
A method is presented to automatically locate a crystal and its holder for centring on a goniometer spindle and alignment with an X-ray beam. Here, a novel algorithm that has been developed and tested with the images of users' crystals saved in an annotated database is described. The algorithm improves on the difficult situations that are commonly observed and poorly handled by the first-generation crystal-centring algorithms. These include highly transparent crystals, bad cryocooling or lens effects arising from the geometry of the drop. Most crystals have polyhedral shapes and a number of straight edges, which yield useful information. In this method, crystal detection relies on a feature-scoring system in which line extraction has the highest weight. Here, the image processing and calculations implemented in the program C3D are described. This program is designed to operate with a client program that controls specific diffractometer hardware. In order to select the best detection conditions, C3D provides various functionalities adapted to various hardware configurations.
Automation , Software , Automation/methods , Crystallography, X-Ray/methods
Structural proteomics has promoted the rapid development of automated protein structure determination using X-ray crystallography. Robotics are now routinely used along the pipeline from genes to protein structures. However, a bottleneck still remains. At synchrotron beamlines, the success rate of automated sample alignment along the X-ray beam is limited by difficulties in visualization of protein crystals, especially when they are small and embedded in mother liquor. Despite considerable improvement in optical microscopes, the use of visible light transmitted or reflected by the sample may result in poor or misleading contrast. Here, the endogenous fluorescence from aromatic amino acids has been used to identify even tiny or weakly fluorescent crystals with a high success rate. The use of a compact laser at 266 nm in combination with non-fluorescent sample holders provides an efficient solution to collect high-contrast fluorescence images in a few milliseconds and using standard camera optics. The best image quality was obtained with direct illumination through a viewing system coaxial with the UV beam. Crystallographic data suggest that the employed UV exposures do not generate detectable structural damage.
Proteins/chemistry , Crystallization , Crystallography , Fluorescence , Insulin/chemistry , Lasers , Muramidase/chemistry , Ultraviolet Rays
The production of three-dimensional crystallographic structural information of macromolecules can now be thought of as a pipeline which is being streamlined at every stage from protein cloning, expression and purification, through crystallisation to data collection and structure solution. Synchrotron X-ray beamlines are a key section of this pipeline as it is at these that the X-ray diffraction data that ultimately leads to the elucidation of macromolecular structures are collected. The burgeoning number of macromolecular crystallography (MX) beamlines available worldwide may be enhanced significantly with the automation of both their operation and of the experiments carried out on them. This paper reviews the current situation and provides a glimpse of how a MX beamline may look in the not too distant future.
Crystallography, X-Ray , Proteins/chemistry , Software , Protein Conformation , Spectrum Analysis/instrumentation , Spectrum Analysis/methods , Synchrotrons/instrumentation
