The TR-icOS setup at the ESRF: time-resolved microsecond UV-Vis absorption spectroscopy on protein crystals.

The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm-2, providing experimental laser and delay parameters for a successful TR-MX experiment.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF. Corrigendum.

A revised version of Table 2 of Nanao et al. [J. Synchrotron Rad. (2022). 29, 581-590] is provided.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF.

ID23-2 is a fixed-energy (14.2 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 (>1013 photons s-1) focused microbeam (minimum size 1.5 µm × 3 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.

ID30A-3 (MASSIF-3) - a beamline for macromolecular crystallography at the ESRF with a small intense beam.

ID30A-3 (or MASSIF-3) is a mini-focus (beam size 18 µm × 14 µm) highly intense (2.0 × 1013 photons s-1), fixed-energy (12.81 keV) beamline for macromolecular crystallography (MX) experiments at the European Synchrotron Radiation Facility (ESRF). MASSIF-3 is one of two fixed-energy beamlines sited on the first branch of the canted undulator setup on the ESRF ID30 port and is equipped with a MD2 micro-diffractometer, a Flex HCD sample changer, and an Eiger X 4M fast hybrid photon-counting detector. MASSIF-3 is recommended for collecting diffraction data from single small crystals (≤15 µm in one dimension) or for experiments using serial methods. The end-station has been in full user operation since December 2014, and here its current characteristics and capabilities are described.

CM01: a facility for cryo-electron microscopy at the European Synchrotron.

Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed in a similar fashion to a synchrotron X-ray beamline. It has been open to the ESRF structural biology user community since November 2017 and will remain open during the 2019 ESRF-EBS shutdown.

ID30B - a versatile beamline for macromolecular crystallography experiments at the ESRF.

ID30B is an undulator-based high-intensity, energy-tuneable (6.0-20 keV) and variable-focus (20-200 µm in diameter) macromolecular crystallography (MX) beamline at the ESRF. It was the last of the ESRF Structural Biology Group's beamlines to be constructed and commissioned as part of the ESRF's Phase I Upgrade Program and has been in user operation since June 2015. Both a modified microdiffractometer (MD2S) incorporating an in situ plate screening capability and a new flexible sample changer (the FlexHCD) were specifically developed for ID30B. Here, the authors provide the current beamline characteristics and detail how different types of MX experiments can be performed on ID30B (http://www.esrf.eu/id30b).

Online Raman spectroscopy for structural biology on beamline ID29 of the ESRF.

Raman spectroscopy can probe the structure and conformations of specific chemical groups within proteins and may thus be used as a technique complementary to X-ray crystallography. This combined approach can be decisive in resolving ambiguities in the interpretation of enzymatic or X-ray induced processes. Here, we present an online Raman setup developed at the European Synchrotron that allows for interleaved Raman spectra acquisition and X-ray diffraction measurements with fast probe exchange and simple alignment while maintaining a high sensitivity over the entire spectral range. This device has been recently employed in the study of a covalent intermediate in the O2-dependent breakdown of uric acid by the cofactor-free enzyme urate oxidase and to monitor its decay induced by X-ray exposure.

RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments.

Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.

MASSIF-1: a beamline dedicated to the fully automatic characterization and data collection from crystals of biological macromolecules.

MASSIF-1 (ID30A-1) is an ESRF undulator beamline operating at a fixed wavelength of 0.969 Å (12.8 keV) that is dedicated to the completely automatic characterization of and data collection from crystals of biological macromolecules. The first of the ESRF Upgrade MASSIF beamlines to be commissioned, it has been open since September 2014, providing a unique automated data collection service to academic and industrial users. Here, the beamline characteristics and details of the new service are outlined.

In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF.

The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for `spectroscopy'). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.

ID29: a high-intensity highly automated ESRF beamline for macromolecular crystallography experiments exploiting anomalous scattering.

ID29 is an ESRF undulator beamline with a routinely accessible energy range of between 20.0 keV and 6.0 keV (λ = 0.62 Što 2.07 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.

The ID23-2 structural biology microfocus beamline at the ESRF.

The first phase of the ESRF beamline ID23 to be constructed was ID23-1, a tunable MAD-capable beamline which opened to users in early 2004. The second phase of the beamline to be constructed is ID23-2, a monochromatic microfocus beamline dedicated to macromolecular crystallography experiments. Beamline ID23-2 makes use of well characterized optical elements: a single-bounce silicon (111) monochromator and two mirrors in Kirkpatrick-Baez geometry to focus the X-ray beam. A major design goal of the ID23-2 beamline is to provide a reliable, easy-to-use and routine microfocus beam. ID23-2 started operation in November 2005, as the first beamline dedicated to microfocus macromolecular crystallography. The beamline has taken the standard automated ESRF macromolecular crystallography environment (both hardware and software), allowing users of ID23-2 to be rapidly familiar with the microfocus environment. This paper describes the beamline design, the special considerations taken into account given the microfocus beam, and summarizes the results of the first years of the beamline operation.