Department of Molecular Carcinogenesis, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
Since the original proof of concept for the utility of an automated procedure for solvent identification and refinement (Tong, H, PhD thesis, 1991), the ASIR procedure has undergone rigorous field tests and proven to be a valuable tool in high resolution protein structure determinations (Tong, H., Berghuis, A.M., Chen, J., Luo, Y., Guss, J.M., Freeman, H.C. & Brayer G.D. 1994. ASIR: an automatic procedure for determining solvent structure in protein crystallography. J. Appl. Cryst. 27, 421-426.). Currently progress is being made in the implemenation of the ASIR approach in conjugation with the widely accepted protein structure analysis program packages CCP4, X-PLOR and TNT. In view of the recent explosive growth of structural biology, the automatic method for solvent structure determination should find an increasingly wider range of applications.
In the test of the ASIR approach in conjugation with CCP4, X-PLOR and TNT, we have used our recently determinated structures of recombinant mouse ubiquitin-conjugating enzyme 9 (mUbc9, Tong, H, Hateboer, Perrakis, A, van Dijk, P., Bernards, R. & Sixma, T.K., in preparation) as a test case. mUbc9 belongs to a family of ubiquitin-conjugating enzymes (E2s) that, together with a family of ubiquitin-ligating enzymes (E3s), are the primary determinants of the specificity of numerous ubiquitin pathways that play critical roles in controlling gene expression, DNA repair, cellular stress response, signal transduction, programmed cell death, cell cycle progression and other vital cellular processes. mUbc9 (Mr = 18 K) is suitable for the test of the ASIR procedure because it crystallized in two different crystal forms (I222: a = 35.39, b = 93.95, c = 115.89; P21: a = 52.36, b = 35.15, c = 57.96, Beta = 111.7) and both types of crystals diffracted to 2.0 Å. Comparison of the mUbc9 solvent structures determined using different program packages in conjugation with ASIR is under way that would provide us with insights into both the scientific and technical aspects of solvent structure determination in protein crystallography, especially with respect to the treatment of disordered water molecules and the identification of solvent molecules other than water.