Towards a high-throughput system for high-pressure cooling of cryoprotectant-free biological crystals

Abstract

A prototype of a high‐pressure cooling apparatus dedicated to macromolecular crystallography on synchrotrons is reported. The system allows cooling of biological crystals without the addition of penetrating or nonpenetrating exogenous cryoprotectant by transforming the aqueous solvent into high‐density amorphous ice at a pressure of 200 MPa. The samples are directly fished from crystallization trays with cryopins specifically designed for the pressurizing device and which are compatible with robotized sample changers on synchrotron beamlines. Optionally, the system allows noble gas derivatization during the high‐pressure cooling procedure. Some technical details of the equipment and of the method are described in this article. A representative series of test crystals shows that the system is capable of successfully cooling samples that normally require a wide variety of cryoprotection conditions. The last section focuses on pressure‐induced structural modifications of these proteins, which are shown to be few but nevertheless of interest.

Tracking the route of molecular oxygen in O 2 -tolerant membrane-bound [NiFe] hydrogenase

“…To investigate whether the pressure treatment, which comes along with the soak-and-freeze method, had an impact on the overall structure of MBH and its gas tunnels, we compared the O 2 -derivatized structure and a structure obtained after high-pressure (2,000 bar of helium) treatment of MBH crystals with the nonderivatized reference structure of MBH at ambient pressure ( 28 ). The crystal structures were aligned using the program PyMOL ( 29 ), and we obtained root mean square deviations (rmsds) of 0.043 Å and 0.195 Å when comparing the O 2 -derivatized structure and the high-pressure structure with the reference MBH structure, respectively ( Fig.…”

“…The cryoprotectant free cooling of the MBH crystals was performed using a high-pressure cooling system recently developed at the ESRF ( 28 ). The needle-shaped crystals were directly harvested from the crystallization drop with specific pluggable sample supports (MiTeGen) and transferred into the pressurizing drop tubes at room temperature.…”

Krypton Derivatization of an O2‐Tolerant Membrane‐Bound [NiFe] Hydrogenase Reveals a Hydrophobic Tunnel Network for Gas Transport

“…Re MBH was produced and purified as described previously . Optimized Re MBH crystals were obtained using sitting‐drop vapor diffusion and were krypton‐pressurized with a high‐pressure cooling system . A detailed description of all experiments can be found in the Supporting Information.…”

Ein Netzwerk aus hydrophoben Tunneln zum Transport gasförmiger Reaktanten in einer O2‐toleranten, membrangebundenen [NiFe]‐ Hydrogenase, aufgedeckt durch Derivatisierung mit Krypton

“…Re MBH wurde wie zuvor beschrieben produziert und gereinigt . Optimierte Re MBH‐Kristalle wurden mit der Dampfdiffusionsmethode im sitzenden Tropfen gezüchtet und in einem Hochdruckkühlungssystem mit Krypton derivatisiert . Eine detaillierte Beschreibung aller Experimente befindet sich in den Hintergrundinformationen.…”