Phases of ~-chymotrypsin (~-CHT) have been determined to 2.8/~ resolution by the multiple isomorphous replacement method using 6 heavy-atom derivatives: (1) Pt( + ), (2) Pt, (3) Hg, (4) U, (5) U + Au, and (6) U(+). The Pt and U derivatives were concentration-dependent and each reagent yielded two useful derivatives while the U + Au derivative was a 'double' derivative. Bijvoet pairs were measured with the U(+) derivative to 5.2 ~ resolution (20< 17 °) in order to determine the absolute configuration of the molecule. Crystals of e-CHT (M.W.~25,300) were grown from half-saturated (NH4)2SO4 at about pH=4.2. The crystals are monoclinic, space group P21, a=49.24 (7), b=67.20 (10), c=65.94 (9)/~,, t= 101.79 (8) °, 4 molecules per cell (2 molecules per asymmetric unit). Intensity data were measured to 2.8 ~ resolution with a computer-controlled diffractometer operating with a crystal alignment feature and using a 'wandering' count 6-drop 2 co step-scan procedure. Reproducibility on the same crystal was about R ~0.02, 0.03 with different crystals and 0.04 for 'before' and 'after' data collections or as a function of extended X-ray exposure (R = Y]I F, I -(I FI)I/E <1FI ), where ]Fit and (I FI ) are the observed and average IFI values, respectively). Two independent sets of intensity data to 3"5 A resolution have been measured on 4 crystals of ~-CHT (two independent preparations). The agreement between the two sets, after data reduction to IFI p's, was R = 0.023 for 4596 independent reflections. Using the average structure amplitudes of the two sets of measurements to 3-5 .~, resolution (4990) and the remainder of the reflections to 2.8 ~ resolution, phase determination and refinement were carried out via alternate cycles of phase calculation and least-squares refinement of heavy-atom parameters minimizing the lack of closure error of the 9044 observed native reflections (85 % of theoretical maximum). The average figure of merit at 2-8/~. resolution is (m)=0.76 (MRE/n=0.50, where n= 6). The average difference between maximum probability and 'best' phase angles is 14.3 °. The r.m.s, calculated structure factors and corresponding lack of closure errors (in parentheses) for the various derivatives are: Pt(+)= 189 (55), Pt = 114 (49), Hg= 104 (58), U=61 (46), U+Au= 104 (46), U(+)= 120 (47) e/u.c.
