Freeze-Trapping Isomorphous Xenon Derivatives of Protein Crystals

A simple and efficient method to prepare isomorphous derivatives of protein crystals with xenon as a heavy atom is described. The method consists of exposing a crystal to xenon gas of pressures above 5 atm (~ 0.5 MPa) for several minutes and subsequently shock-freezing the crystal to immobilize the xenon dissolved in the mother liquor and bound to the protein. Diffraction data can the be collected with the established techniques of protein cryocrystallography. Two types of high-pressure device are described to expose a protein crystal to the required xenon pressure, permitting rapid freeze-quenching after xenon exposure. One of these devices can be used for gas pressures up to and exceeding 5.0 MPa, with a gas consumption of a few millilitres of uncompressed gas. The technique has been tested with monoclinic crystals of sperm-whale metmyoglobin, which has four xenon binding sites. The results of these experiments are described and discussed. Potential applications of this technique include-besides the classical multiwavelength anomalous diffraction (MAD) or single isomorphous replacement with anomalous scattering (SIRAS) experiments-the derivation of low-angle phase information by modifying the electron density of the solvent regions within the crystal.