THE SOLVENT STRUCTURE OF CONCANAVALIN A ANALYSED AT 2Å AND THEN 0.94Å RESOLUTION. T. Gleichmann, A. Deacon, S.Trapani and J. R. Helliwell, Department of Chemistry, University of Manchester M13 9PL, UK

Our investigation deals with the reliability and verification of water molecules in proteins found by X-ray crystallography at both room temperature (to 2Å resolution) and low temperature (0.94 Å resolution). At room temperature several 2Å coordinate sets of concanavalin A were used as test cases to determine the number of conserved sites ie within the solvent structures of different crystal packing arrangements of the sugar free and two distinct sugar bound forms of the protein. Within the sugar free crystal form it was found that three waters in the sugar binding site are conserved in a number of derivatives (Ni, Co, Mn, Cd substituted concanavalin A) and match the positions of three sugar-oxygen atoms (from the sugar bound crystal structures). Overall in the three different crystal packings a large number of solvent sites are conserved; within a distance range of 0 to 1.2Å, 73 water sites (49%) in a mannoside subunit and 80 water sites (54%) in a glucoside subunit are conserved compared to the cobalt-substituted sugar free concanavalin A subunit (at 1.6Å, the best room temperature crystal structure coordinate set we have). In order to assess methods' aspects of the reliability and precision of the water structure we also compared two Co-concanavalin A structures at room temperature.

One was obtained with Laue data (at 2Å) and the other was with the 1.6Å monochromatic data set. A total of 150 waters were found from the Laue refinement of which 119 agreed with the monochromatic refinement (including the three receptor binding site waters). Finally by use of of flash freezing, a short wavelength intense SR beam (CHESS) and a CCD area detector a resolution of 0.94 A has been achieved for the sugar free crystal form (this is a record for a protein of 25kDa molecular weight, to our knowledge!). This X ray data set comprises 117000 unique reflections (75.4% complete). In this low-temperature refined structure (R-factor, SHELXL93, 13.1%) the number of detected solvent sites has increased from 147 to 290 and the quality of the electron density greatly enhanced.