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Title
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Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP.
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Authors
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M.Del Campo,
A.M.Lambowitz.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2009,
65,
832-835.
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PubMed id
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Abstract
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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/Delta598-664) were
overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited
low solubility in standard solutions (< or =1 mg ml(-1)), but its solubility
could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50%
glycerol to achieve concentrations of approximately 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/Delta598-664 in complex with AMP-PNP and U(10) belonged to space group
P2(1)2(1)2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 A, and
diffracted X-rays to beyond 1.9 A resolution using synchrotron radiation from
sector 21 at the Advanced Photon Source.
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