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.