Open AccessExpression, purification, crystallization and preliminary X‐ray diffraction analysis of the DDX3 RNA helicase domain
Author(s) -
Rodamilans Bernardo,
Montoya Guillermo
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309107006434
Subject(s) - helicase , rna helicase a , crystallography , ammonium sulfate , crystallization , dead box , monoclinic crystal system , rna , chemistry , biochemistry , crystal structure , gene , chromatography , organic chemistry
DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD‐box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp‐Glu‐Ala‐Asp motif that defines the family. DDX3 has two distinct domains: an ATP‐binding domain in the central region of the protein and a helicase domain in the carboxy‐terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli . Crystallization experiments yielded crystals that were suitable for X‐ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 m M spermine tetrahydrochloride and a protein solution containing 10 m M HEPES, 500 m M ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P 2 1 , with unit‐cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).