Materials and Methods

Thermolysin was obtained from Calbiochemand crystallized as described by Holmes & Matthews (1982). The crystals were stored in a mother liquor of 0.01 M calcium acetate, 0.01 M Tris-acetate, and 7%(v/v) dimethyl sulfoxide, pH 7.2. Complexes of thermolysin with the inhibitor CLT, a gift of Dr. AA Patchett (Maycock et al., 1981), were obtained by soaking native crystals in mother liquor containing the inhibitor. The amount of binding was monitored by calculating (. hOl) difference Fourier projections [cf. Weaver et al.(1977)]. A single thermolysin crystal soaked for 12 days with 50/uM CLT was used for three-dimensional data collection. Data were collected to 1.7-A resolution by use of oscillation photography (Rossmann, 1979; Schmid et al., 1981). Thermolysin crystals generally grow as hexagonal rods, enabling us to make 12 exposures at one end of the crystal and then to translate the crystal horizontally to an unexposed portion, which was used for the remaining 16 exposures. An oscillation angle of 1.2 per film pack was used, and the crystals (space group P (> i22) were rotated about the c axis through a net rotation of 30. The X-ray source was a graphite-monochromatized Elliot GX-21 rotating anode generator operated at 39 kV, 130 mA, and the exposure time was 6 h per film. Intensity sta-tistics are summarized in Table I.