CRYSTALLIZATON AND REFINED STRUCTURE OF RIBONUCLEASE S COMPLEXED WITH A SUBSTRATE ANALOG. Igor V. Pechik¹ and Gary L. Gilliland². ¹V.A.Engelhardt Institute of Molecular Biology Russian Academy of Sciences, 32 Vavilov St., Moscow, 117984,Russia. ²Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and National Institute of Standards and Tech-nology, 9600 Gudelsky Dr., Rockville, MD 20850,USA

A novel procedure was developed to crystallize the complex of RNase S with UpcA, an analog of di-nucleotide substrate UpA that has the 5' oxygen substituted with a methylene group. The previous procedure for crystallizing RNase S was based on the use of high concentrations of ammonium sulfate [Wyckoff et al., (1967) J.Biol.Chem., 242, 3749 - 3753]. The modified conditions for crystal growth use only 22-26% saturated ammonium sulfate which is more favourable for UpcA binding, and 50% saturated sodium chloride in 0.1 M sodium acetate buffer at pH 5.0. The concentration of RNase S in droplets was 30 mg/ml with its 1:10 ratio to UpcA [Gilliland et al., (1994) Protein and Peptide Letters, v.1, 60 - 65]. The vapor diffusion procedure appears to be highly stable and reproducable, producing single crystals of the average size about 0.7-1.0 mm. X-ray diffraction data from the unliganded enzyme and its complex with UpcA were collected to 1.8 A resolution with the completeness of 98%.Both struc- tures were refined with final R-factors of 0.167 and 0.17 respectively. Some parts of UpcA molecule can be seen only at the low level of the electron density implying their high mobility. The structure of the complex will be described and compared with the unliganded enzyme. The results of the analysis that focus on structural changes of the protein induced by interactions with the ligand at both the B1 and B2 sites, differences in solvent structure, and the role that water molecules play in the ligand recognition and binding will be presented.