POLIOVIRUS 3C PROTEASE CRYSTALLIZATION AND STRUCTURE SOLUTION. S. C. Mosimann1, M. Chernaia1, S. Plotch2, Y. Gluzman2, M. N. G. James1, 1MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Canada TOG 2H7, 2Wyeth Ayerst Research, Division of American Home Products, Pearl River, New York, 10965
The 3C gene produce of poliovirus is a cysteine protease required for vira1 infectivity[l]. The 3C protease is responsible for the bulk of the processing of the viral polyprotein into its individual proteins[2]. Recombinant poliovirus 3C protease has been expressed in E. coli, solubilized from inclusion bodies and purified to homogeneity. Large single crystals of poliovirus 3C protease have been grown from hanging drops containing (NH4)2SO4 solutions. The crystals are orthorhombic (space group P212121), the unit cell parameters are a=78.95Å, b=116.30Å, c=47.14Å, [[alpha]]=[[beta]]=[[gamma]]=90deg. and there are two molecules of 3C protease in the asymetric unit. The calculated solvent content is 55% and the specific volume is 2.64Å3/Da[2]. A complete 2.5Å X-ray intensity data set (Rmerge=0.052) has been collected using monochromated CuK[[alpha]] radiation from a rotating anode source and San Diego Multiwire Systems proportional counters. To date, one exceptional isomorphous derivative (uranyl acetate) has been identified and electron density maps calculated with the SIR phases clearly reveal the two molecules in the asymmetric unit. Solvent flattening and histogram matching using the program DM[3] have significantly improved the quality of the SIR electron density maps. The anomalous signal from the uranyl acetate derivative and the presence of right-handed [[alpha]]-helices have been used to confirm the correct hand of the SIR density maps. At present, the poliovirus 3C protease sequence is being fit to the SIR electron density maps.
[l] Baum et al, Virology 185, 140 (1991)
[2] Ypma-Wong et al., Virology 166, 265 (1988)
[3] Cowtan, Joint CCP4 & ESF-EACBM Newsletter 31, 34 (1994)
[4] Matthews, J. MoL Biology 33, 491 (1968)