Abstract.-Independent 4 A electron density maps calculated for the extracellular nuclease of Staphylococcus aureus (based on data from three heavy-atom derivatives) and for a nuclease-thymidine-3',5'-diphosphate-calcium ion complex (based on a single isomorphous derivative) show about 60 per cent of the chain resolved, including 31/2 turns of helix. The pyrimidine ring of the inhibitor fits into a pocket in the enzyme and appears to be parallel to the ring of a tyrosyl residue. Conformational changes can be observed between the nuclease and the nuclease-inhibitor complex, but the two structures seem to be identical over most of the molecule.The extracellular nuclease of Staphylococcus aureus, a calcium ion-activated enzyme that attacks the phosphodiester bonds of both RNA and DNA, consists of a single chain of 149 amino acid residues (mol. wt. = 16,807) with no sulfhydryl or disulphide groups. C. B. Anfinsen and his associates have recently reviewed1 their extensive work on the chemistry of this nuclease, including a sequence determination and progress toward total synthesis. We previously reported the crystallization of the nuclease in several forms2; the present paper describes our further progress with an X-ray crystallographic determination of the structure.Methods and Results.-Nuclease (Foggi strain) was purchased from Worthington Biochemical Corp. Used material was recovered and preparations that gave poor crystals were reworked, originally by recrystallization, but more recently by chromatography on phosphocellulose.3 As previously described,2 uninhibited nuclease crystals were grown in siliconed vials at 20C from a 1-2 mg/ml solution of nuclease in pH 8.15, 0.0105 M potassium phosphate buffer with 2-methyl-2,4-pentanediol4 added dropwise to a final concentration of 29-32 per cent and a final pH (at 250) of 8.6-8.7. Good crystals formed in 1-2 months; they were tetragonal, space group P41, of unit cell dimensions a = 47.75, c = 63.5 A. For nuclease-inhibitor crystals, a solution of enzyme and buffer (as above) with 22-25 per cent diol was allowed to stand at 20C for several days and then filtered through a 0.45 As pore-size i\iillipore filter in a filtering centrifuge tube. One mole per mole nuclease of thymidine-3',5'-diphosphate (pdTp), a potent inhibitor,' was added, and two moles Ca2+ in the form of 0.01 I\{ calcium chloride-0.02 1\1 potassium citrate were also added. These crystals grew more rapidly than the native nuclease crystals and were of the same space group but with unit cell dimensions of a = 48.3, c = 63.3 A. No crystals of either type that varied more than 0.1 A from the given unit cell dimensions were used. Crystal size ranged from 0.15 X 0.15 X 0.5 mm for some nuclease-pIdUp crystals to 0.4 X 0.5 X 0.7 420

No abstract