THE STRUCTURES OF TWO RIBONUCLEASE B CONTAINING CRYSTALS (X-RAY CRYSTALLOGRAPHY, RNA)

Ribonuclease B was crystallized in a C2 unit cell with parameters a = 101.81 A., b = 33.36 A., c = 73.60 A. and beta = 89.6 degrees. The crystal had two protein molecules per asymmetric unit. The arrangement of the molecules within the asymmetric unit was determined using molecular replacement methods and subsequently refined using reciprocal space least-squares procedures. The R factor for the 2.8 A. refinement was .23 and the overall deviation from ideal geometry was .049. The correctness of the structure was confirmed with difference Fourier syntheses using the data obtained for three heavy atom derivatives: rutheninum pentaamine chloride, platinum chloride, and 5-iodouridine. In agreement with studies from other laboratories, the ruthenium pentaamine was found near His 105, the platinum was found bound to Met 29 of both molecules in the asymmetric unit, and the 5-iodouridine was found in the pyrimidine sites of both molecules. Upon examination of the intermolecular contacts, it was found that the contacts around the dyad axes were identical to those reported for the P31212 and C2 crystals of ribonuclease S. This preserved dimeric interaction is stabilized by a salt link between Arg 85 and Asp 121.

Ribonuclease B was also co-crystallized with tetradeoxyadenylate (DA4) yielding crystals in space group P41212 with dimensions a = b = 44.45 A. and c = 156.5 A. The orientation and position of the molecule in the asymmetric unit was determined by molecular replacement. The protein structure was partially refined to an R factor of .35 to 3.0 A. The correctness of the structure was ascertained by examining a difference Fourier synthesis for a platinum chloride derivative. It was found that platinum bound to sulphur of Met 29. A difference Fourier synthesis was also calculated using /Fobs - Fcalc/ coefficients in order to find the DNA in the crystal. A strong density was found associated with the active site in a manner similar to what was observed for a tetradeoxyadenylate (DA4) molecule reported in the P22121 crystalline complex of ribonuclease A and DA4. The protein-protein contacts around one of the screw axes in the P41212 unit cell are identical to that found around one of the screw axes in the P212121 complex.