CRYSTALLIZATION OF A FRAGMENT OF HUMAN FIBRONECTIN - INTRODUCTION OF METHIONINE BY SITE-DIRECTED MUTAGENESIS TO ALLOW PHASING VIA SELENOMETHIONINE

Crystals of a fragment of human fibronectin encompassing the 7th through the ROD-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 Angstrom 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. (C) 1994 Wiley-Liss, Inc.