Crystallization and preliminary X-ray analysis of the CRP–cAMP–DNA (full length) complex

Abstract

The Escherichia coli cyclic AMP receptor protein (CRP) is a well known transcription activator protein. In this study, CRP was overexpressed, purified and cocrystallized with cAMP and a 38 bp full-length double-stranded DNA fragment. The full-length segment differed from the half-site fragments used in previous crystallization experiments and is more similar to the environment in vivo. CRP-cAMP-DNA crystals were obtained and diffracted to 2.9 Å resolution. The crystals belonged to space group P3 1 21, with unit-cell parameters a = b = 76.03, c = 144.00 Å . The asymmetric unit was found to contain one protein molecule and half a 38 bp full-length double-stranded DNA fragment, with a Matthews coefficient of 2.62 Å 3 Da À1 and a solvent content of 53.14%.

Revisiting the mechanism of activation of cyclic AMP receptor protein (CRP) by cAMP in Escherichia coli: Lessons from a subunit‐crosslinked form of CRP

“…The exact organization of the DNA binding domain in the CRP-cAMP-DNA complex can only be visualized by obtaining the structure of a CRP-cAMP-DNA complex using a DNA fragment containing the full binding site of CRP. Crystallization of such a complex has recently been reported [24], but the solved structure is not yet available. Table 1 S-S distance between the C178 residues of the two subunits in different CRP structures.…”

Crystallization and preliminary X-ray diffraction analysis of D53H mutantEscherichia colicAMP receptor protein

“…The D53H CRP mutant was expressed and purified using the same protocol as used for the wild type in a previous study (Huang et al, 2013). The cells were resuspended in 250 ml buffer A (50 mM Tris-HCl, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 5% glycerol pH 7.8) and lysed by pressure (JN-3000 Plus, JNBIO, People's Republic of China) at 277 K. The cell lysate was centrifuged at 15 000 rev min À1 and 277 K for 30 min.…”