Purification, crystallization and preliminary crystallographic data of the m3G cap-binding domain of human snRNP import factor snurportin 1.
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 A, alpha = beta = gamma = 90 degrees. Crystals contain one molecule in the asymmetric unit and diffract to a resolution limit of 2.9 A.
Strasser
Anja
A
Abteilung Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Justus-von-Liebig Weg 11, 37077 Göttingen, Germany.
Dickmanns
Achim
A
Schmidt
Ute
U
Penka
Elke
E
Urlaub
Henning
H
Sekine
Mitsuo
M
Lührmann
Reinhard
R
Ficner
Ralf
R
D003432:Cross-Linking Reagents; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D039502:Nuclear Cap-Binding Protein Complex / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D039381:RNA Cap-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D039502:Nuclear Cap-Binding Protein Complex; D009841:Oligonucleotides; D010446:Peptide Fragments; D039381:RNA Cap-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C114090:SNUPN protein, human