HIGH-PRESSURE KRYPTON GAS AND STATISTICAL HEAVY-ATOM REFINEMENT - A SUCCESSFUL COMBINATION OF TOOLS FOR MACROMOLECULAR STRUCTURE DETERMINATION

The noble gas krypton is shown to bind to crystallized proteins in a s imilar way to xenon [Schiltz, Prange & Fourme (1994). J. Appl. Cryst. 27, 950-960]. Preliminary tests show that the major krypton binding si tes are essentially identical to those of xenon. Noticeable substituti on is achieved only at substantially higher pressures (above 50 x 10(5 ) Pa). As is the case for xenon, the protein complexes with krypton ar e highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypt on is not as heavy as xenon, but its K-absorption edge is situated at a wavelength (0.86 Angstrom) that is readily accessible on synchrotron radiation sources. As a test case, X-ray diffraction data at the high -energy side of the K edge were collected on a crystal of porcine panc reatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 x 10(5) Pa. The occupancy of the single Kr atom is app roximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used success fully for phase determination with the SIRAS method (single isomorphou s replacement with anomalous scattering). After phase improvement by s olvent flattening, the resulting electron-density map is of exceptiona lly high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomo rphous and anomalous signals have proven to be crucial in the determin ation of this electron-density map. Heavy-atom refinement and phasing were carried out with the program SHARP, which is a fully fledged impl ementation of the maximum-likelihood theory for heavy-atom refinement [Bricogne (1991). Crystallographic Computing 5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257-297. Oxford: Clarendon Press]. I t is concluded that the use of xenon and krypton derivatives, when the y can be obtained, associated with statistical heavy-atom refinement w ill allow one to overcome the two major limitations of the isomorphous replacement method i.e. non-isomorphism and the problem of optimal es timation of heavy-atom parameters.