The Crystal Structure of Myoglobin. IV. A Fourier Projection of Sperm-Whale Myoglobin by the Method of Isomorphous Replacement

Physics

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Scientific paper

Two methods were used to attach heavy atoms to specific sites on the myoglobin molecule in type A crystals: (i) the use of specific reagents for the haem group, including isocyanides and nitroso compounds, (ii) the crystallization of the protein in the presence of various ions, especially mercuri-iodide, aurichloride, mercury diammine, and p-chloro-mercuri-benzene sulphonate. The first method was not completely successful, largely because most of the reagents were so readily displaced by oxygen: the haem group was, however, located by this means. By the second method isomorphous replacements were successfully achieved at four different sites; the chemical mechanism of attachment is in no case well understood. The classical method of isomorphous replacement was used to determine the signs of the h0l (real) reflexions out to spacings of 4 angstrom. x- and z-co-ordinates of the heavy atoms were found by computing difference-Patterson projections, and the signs of the protein reflexions were deduced in the usual manner by relating the signs of the calculated heavy-atom structure factors to the changes of amplitude caused by the introduction of the heavy atom. As a cross-check, signs were independently determined from each of the isomorphous replacements in turn. An electron-density projection of the protein along y was computed; it included all terms of spacing greater than 4 angstrom, but could not be interpreted in terms of chemical structure owing to the degree of overlap inevitable in such a projection (the axis of projection being 31 angstrom). However, the relative positions of the two protein molecules in the unit cell were established by means of a salt-water difference-Fourier projection. To obtain further information about the structure of the molecule it will be necessary to work in three dimensions; it was with this end in view that so large a variety of isomorphous replacements was investigated.

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