Gas-sensitive biological crystals processed in pressurized oxygen and krypton atmospheres: deciphering gas channels in proteins using a novel `soak-and-freeze' methodology

CERVOXY COLLInternational audienceMolecular oxygen (O2) is a key player in many fundamental biological processes. However, the combination of the labile nature and poor affinity of O2 often makes this substrate difficult to introduce into crystals at sufficient concentrations to enable protein/O2 interactions to be deciphered in sufficient detail. To overcome this problem, a gas pressure cell has been developed specifically for the `soak-and-freeze' preparation of crystals of O2-dependent biological molecules. The `soak-and-freeze' method uses high pressure to introduce oxygen molecules or krypton atoms (O2 mimics) into crystals which, still under high pressure, are then cryocooled for X-ray data collection. Here, a proof of principle of the gas pressure cell and the methodology developed is demonstrated with crystals of enzymes (lysozyme, thermolysin and urate oxidase) that are known to absorb and bind molecular oxygen and/or krypton. The successful results of these experiments lead to the suggestion that the soak-and-freeze method could be extended to studies involving a wide range of gases of biological, medical and/or environmental interest, including carbon monoxide, ethylene, methane and many others

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

International audienceRecent 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

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

International audienceThe 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

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

International audienceAutomation 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 automaticall

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

International audienceID30A-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