A decade of user operation on the macromolecular crystallography MAD beamline ID14-4 at the ESRF

The improvement of the X-ray beam quality achieved on ID14-4 by the installation of new X-ray optical elements is described

The ID23-2 structural biology microfocus beamline at the ESRF

Beamline ID23-2, the first dedicated and highly automated high-throughput monochromatic macromolecular crystallography microfocus beamline, is described

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments

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

Le rayonnement synchrotron : comprendre la relation structure-fonction des macromolécules biologiques

Ces trois dernières décennies ont vu un bouleversement complet de la biologie structurale grâce à l’utilisation du rayonnement synchrotron. De nombreuses structures de macromolécules biologiques, inconnues jusqu’à ce jour, ont ainsi été déterminées. L’automatisation des mesures de diffraction et le développement des techniques d’analyse et de modélisation ont facilité l’exploitation de ce nouvel outil. Ces études structurales ont ainsi pu donner accès aux détails du fonctionnement des protéines et des molécules médicamenteuses qui interagissent avec elles, par exemple dans les combats contre les cancers ou le VIH, et ont influencé le développement de nouvelles thérapies

Fully automatic macromolecular crystallography: the impact of MASSIF-1 on the optimum acquisition and quality of data

International audienceAutomation is beginning to transform the way data are collected in almost all scientific disciplines. The combination of robotics and software now allows data to be collected consistently and reproducibly, eliminating human error and boredom. This approach has been applied to macromolecular crystallography at MASSIF-1, a fully automated beamline at the European Synchrotron Radiation Facility (ESRF). Considerable human effort is still dedicated to evaluating protein crystals in order to find the few crystals that diffract well or collecting hundreds of data sets to screen potential new drug candidates. The combination of ESRF-developed robotic sample handling and advanced software protocols now provides a new tool to structural biologists. Not only is the beamline used efficiently, running 24h a day without getting tired, data collection is also performed consistently by an expert system, often better than with a human operator. In this review, we will focus on the impact this level of automation has had on the optimum acquisition of data from crystals of biological macromolecule

Recent progress in robot-based systems for crystallography and their contribution to drug discovery.

International audienceX-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein-ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms. The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline. The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community

Fully automatic characterization and data collection from crystals of biological macromolecules

International audienceConsiderable effort is dedicated to evaluating macromolecular crystals at synchrotron sources, even for well established and robust systems. Much of this work is repetitive, and the time spent could be better invested in the interpretation of the results. In order to decrease the need for manual intervention in the most repetitive steps of structural biology projects, initial screening and data collection, a fully automatic system has been developed to mount, locate, centre to the optimal diffraction volume, characterize and, if possible, collect data from multiple cryocooled crystals. Using the capabilities of pixel-array detectors, the system is as fast as a human operator, taking an average of 6min per sample depending on the sample size and the level of characterization required. Using a fast X-ray-based routine, samples are located and centred systematically at the position of highest diffraction signal and important parameters for sample characterization, such as flux, beam size and crystal volume, are automatically taken into account, ensuring the calculation of optimal data-collection strategies. The system is now in operation at the new ESRF beamline MASSIF-1 and has been used by both industrial and academic users for many different sample types, including crystals of less than 20 mu m in the smallest dimension. To date, over 8000 samples have been evaluated on MASSIF-1 without any human interventio

Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa

Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d(1) heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described [Fulop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412], and we recently reported on the structure of oxidized NiR-Pa at 2.15 Angstrom [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Although the domains carrying the d(1) heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d(1) heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd. Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d(1) heme active site. Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 Angstrom away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse