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

Daniel J. Leahy, Harold P. Erickson, Ikramuddin Aukhil, Paritosh Joshi, Wayne A. Hendrickson

Research output: Contribution to journalArticle

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. © 1994 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)48-54
Number of pages7
JournalProteins: Structure, Function, and Bioinformatics
Volume19
Issue number1
DOIs
StatePublished - May 1994

Keywords

  • X‐ray crystallography
  • extracellular matrix
  • multiwavelength anomalous diffraction (MAD)

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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