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 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 A 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.
Leahy
D J
DJ
Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York 10032.
Erickson
H P
HP
Aukhil
I
I
Joshi
P
P
Hendrickson
W A
WA
D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012645:Selenomethionine
D005353:Fibronectins; D010446:Peptide Fragments; D012645:Selenomethionine; D008715:Methionine