Crystallization of a fragment of human fibronectin: Introduction of methionine by site‐directed mutagenesis to allow phasing via selenomethionine

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Abstract
Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7–10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 Å Bragg spacings. A mutant FN7–10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.