Open AccessCrystallization and preliminary X‐ray diffraction of the DEAD‐box protein Mss116p complexed with an RNA oligonucleotide and AMP‐PNP
Author(s) -
Del Campo Mark,
Lambowitz Alan M.
Publication year - 2009
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309109027225
Subject(s) - oligonucleotide , crystallization , rna , crystallography , x ray , x ray crystallography , diffraction , chemistry , microbiology and biotechnology , materials science , dna , biochemistry , biology , physics , gene , organic chemistry , optics
The Saccharomyces cerevisiae DEAD‐box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA‐end processing. For crystallization trials, full‐length Mss116p and a C‐terminally truncated protein (Mss116p/Δ598–664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (≤1 mg ml −1 ), but its solubility could be increased by adding 50 m M l ‐arginine plus 50 m M l ‐glutamate and 50% glycerol to achieve concentrations of ∼10 mg ml −1 . Initial crystals were obtained by the microbatch method in the presence of a U 10 RNA oligonucleotide and the ATP analog AMP‐PNP and were then improved by using seeding and sitting‐drop vapor diffusion. A cryocooled crystal of Mss116p/Δ598–664 in complex with AMP‐PNP and U 10 belonged to space group P 2 1 2 1 2, with unit‐cell parameters a = 88.54, b = 126.52, c = 55.52 Å, and diffracted X‐rays to beyond 1.9 Å resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.