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X‐ray crystallographic study of binding of cobalt ion to hen egg‐white lysozyme
Author(s) -
Yonath Ada,
Smilansky Anita,
Sharon Nathan
Publication year - 1974
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(74)80506-5
Subject(s) - white (mutation) , chemistry , lysozyme , citation , library science , crystallography , computer science , biochemistry , gene
The present studies were carried out essentially as described by Moult et al. [3]. Tetragonal crystals of hen egg-white lysozyme [4] were grown at pH 4.7 in 0.02 M acetate buffer, containing 5% NaCl (w/v). Individual crystals (space group P4s2r2, a=b=79.1 A, C= 37.9 A) about 0.6 mm in the longest dimension, were transferred to 1 .O mm diameter quartz capillaries containing 0.1 ml mother liquor. About 2 mg of solid CoS04 was added to the capillary to obtain an approximate molar ratio of 1: 100 lysozyme to CoSO+ Soaking was done at room temperature for 3-4 days. The excess of mother liquor was removed from the capillary with a syringe and filter paper. Precession photographs (/J= 18”, minimum spacing=25 A) of the hko, ok1 and hhl centrosymmetric zones, were taken with two films in a pack and exposures of 40-45 hr, using a copper Xray tube running at 40 kV and 28 mA and a nickel filter. The maximum exposure time for a crystal was 55 hr. The crystals were isomorphous with those of native lysozyme and showed only small changes in intensities. A microdensitometer (Optronics photoscan P-1000) wa: used for complete recording of the diffraction patterns, and the integrated intensities of the reflections were obtained by processing on an IBM 370/165 computer, after corrections for background, and for Lorenz and polarization factors. Intensities of equivalent reflections within a pack were scaled together, and compared to the corresponding data for the native crystals. Difference Fourier projections were calculated using the amplitudes of native lysozyme (D. C. Phillips, personal communication) and of Co++-lysozyme, with phases of the native enzyme, and weights based on the native figures of merit.