PROXIMA-1 beamline for macromolecular crystallography measurements at Synchrotron SOLEIL.

The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled its first users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8 h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to 1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via `mail-in' services or remotely.

MXCuBE2: the dawn of MXCuBE Collaboration.

MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.

Energy resolution of the CdTe-XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals.

The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL. The X-ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging-plate detector and then measured with both the XPAD3S-CdTe and the XPAD3S-Si, a silicon photon-counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit-cell parameter determination and the measurement of high-quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X-ray diffraction opened up by this type of technology are discussed.

Evidence for conformational changes upon copper binding to Cupriavidus metallidurans CzcE.

CzcE is a periplasmic protein from Cupriavidus metallidurans CH34 that can bind four copper atoms per dimer. We have crystallized the apo form of the protein and determined its structure at 1.85 A resolution. Three Cu atoms were localized by soaking apo-CzcE crystals into a CuCl(2) solution. We identified His24 as a Cu(II) ligand in each protomer and Asp100 as a key residue for Cu binding at the interface of the dimer. The role of these amino acids was confirmed by site-directed mutagenesis and UV-visible spectroscopy. The fourth Cu atom was not located. The oxidized form of CzcE contains four Cu(II) atoms, while the reduced form contains four Cu(I) atoms. Average coordination spheres of four N or O atoms for Cu(II) and of one N or O atom and two S atoms for Cu(I) were determined by X-ray absorption spectroscopy. As there is no evidence for preformed metal-binding sites in apo-CzcE, we suggest that different conformational changes occurred upon Cu(II) or Cu(I) binding. These changes were further demonstrated by digestion experiments that gave different proteolysis patterns depending not only on the presence of the metal but also on its speciation. The ability of CzcE to bind copper and to adapt its conformation to different copper oxidation states could be related to a role in copper sensing for this protein.

Instrumentation for synchrotron-radiation macromolecular crystallography.

X-ray diffraction is an extremely important tool for structure determination of biological macromolecules, to the extent that currently around 85% of Protein Data Bank entries result from X-ray measurements. Many of these structure determinations use synchrotron radiation for data collection. This article aims to give synchrotron users an overview of the functioning of a synchrotron beamline and how the performance of various instruments combines to allow the collection of diffraction data.