Purification, crystallization and preliminary crystallographic data of the m(3)G cap-binding domain of human snRNP import factor snurportin 1

Abstract

The nuclear import of spliceosomal UsnRNPs is mediated by the transport adaptor snurportin 1 (SPN1), which specifically recognizes the 2,2,7-trimethylguanosine (m(3)G) cap at the 5' end of UsnRNAs. Human SPN1 was overexpressed as a GST-fusion protein in Escherichia coli and purified to homogeneity Since full-length SPN1 did not crystallize, limited proteolysis experiments were performed and stable digestion products were analyzed for functionality with respect to m(3)G cap-binding activity and subsequently used for crystallization trials. Well diffracting single crystals of a truncated SPN1 m(3)G cap-binding domain (residues 79-300) were obtained after two rounds of seeding. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 57.47, c = 130.09 Angstrom, alpha = beta = gamma = 90degrees. Crystals contain one molecule in the asymmetric unit and diffract to a resolution limit of 2.9 Angstrom.