title: Purification, crystallization and preliminary X-ray crystallographic analysis of a central domain of human splicing factor 1.
abstract: Pre-mRNA splicing is an essential source of genetic diversity in eukaryotic organisms. In the early stages of splicing, splicing factor 1 (SF1) recognizes the pre-mRNA splice site as a complex with its partner, U2 auxiliary factor 65 kDa subunit (U2AF(65)). A central `mystery' domain of SF1 (SF1md) lacks detectable homology with known structures, yet is the region of highest phylogenetic sequence conservation among SF1 homologues. Here, steps towards determining the SF1md structure are described. Firstly, SF1md was expressed and purified. The presence of regular secondary structure was verified using circular dichroism spectroscopy and the SF1md protein was then crystallized. A native data set was collected and processed to 2.5 Å resolution. The SF1md crystals belonged to space group C2 and have most probable solvent contents of 64, 52 or 39% with three, four or five molecules per asymmetric unit, respectively. Mutually perpendicular peaks on the κ = 180° section of the self-rotation function support the presence of four molecules in the asymmetric unit.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D000072260:RNA Splicing Factors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D004268:DNA-Binding Proteins; D000072260:RNA Splicing Factors; C107467:SF1 protein, human; D014157:Transcription Factors