Nucleic Acids Research, 2009, Vol. 37, No. 14 4547 Table 1. Diﬀraction data and reﬁnement statistics for the regulatory domain of CrgA SeMet RD-CrgA Data collection X-ray source Detector Space group Unit-cell parameters a (A˚ ) b (A˚ ) c (A˚ ) b (8) Wavelength (A˚ ) Resolution range (A˚ ) Last resolution shell (A˚ ) Unique reﬂections Completeness (%) Redundancy Average I/s (I) Rmerged d’’/se Reﬁnement Resolution (A˚ ) No of reﬂections Rwork/Rfree Number of atoms Protein Water Other Rmsd bonds (A˚ ) RMSD angles (8) B Wilson (A˚ 2) Mean B factor Protein Water Other Ramachandran plot Most favored (%) Allowed (%) I Peak BM14 MAR225 CCD P21 51.59 65.49 63.31 105.27 0.9780 50.0–2.76 2.86 –2.76 10 553 (1058) 99.9 (99.5) 7.4 (6.6) 20.1 (4.2) 0.108 (0.422) 2.48 (1.27) Remote Inﬂection 0.9065 8620 (235) 80.8 (22.1)a 4.0 (1.5) 14.1 (3.3) 0.097 (0.286) 1.38 (0.87) 0.9785 8096 (167) 75.9 (15.7)b 3.5 (1.3) 13.2 (3.8) 0.097 (0.214) 1.38 (0.91) II Peak BM14 MAR225 CCD P21 51.64 65.58 63.50 105.02 0.9795 50.0–2.29 2.37–2.29 15 951 (636) 85.8 (34.8)c 4.1 (1.8) 18.0 (2.1) 0.073 (0.299) 30–2.30 14 781 20.1/25.8 3139 203 2 0.006 1.0 34 30.7 30.5 33.2 39.6 99.5 0.5 Values in parentheses are for data in the highest resolution shell. a,b,cData were processed into the corners of the detector and are essentially complete to 3.5 A˚, 3.8 A˚, 2.7 A˚, respectively. d Phkl Pi | Ii(hkl) –hI(hkl) |/Phkl Pi Ii(hkl). eARnmoermgea=lous signal/noise ratio as calculated by SHELXC, with data from resolution 50–8.0 A˚ and 5–4.2 A˚ respectively. Bioinformatics and structure analysis Structural neighbors of CrgA were identiﬁed using Dali (31). To ﬁnd suitable structures for modeling the interac- tion of CrgA with DNA, all the structures in the Protein Data Bank solved by X-ray crystallography containing both protein and DNA were selected for a pair-wise pro- tein structural comparison using secondary-structure matching (SSM) (32). The interface properties were ana- lyzed using protein interfaces, surfaces and assemblies service PISA at European Bioinformatics Institute (33). The residue conservation of CrgA was calculated using Consurf (34). The eﬀector pocket was analyzed with the Computed atlas of surface topography of proteins CASTp (35). The structural ﬁgures were produced using Pymol (36). Preparation of CrgA, CrgA–DNA complex and EMSA CrgA protein was prepared as previously described in 20 mM Tris pH7.5, 200 mM NaCl (20). DNA of the 63-bp CrgA sequence footprint (ACTTTATAATTTAA AAGTG CAAAAATAAGAAAACACTTTTGCGTCA AATGAAATAATCAGATG), the 30-bp CrgA site 1 (ACTTTATAATTTAAAAGTGCAAAAATAAGA) the 30-bp CrgA site 2 (CACT TTTGCGTCAAATGAAAT AATCAGATG) (18) and the 32-bp control DNA (AAA TTTTCAAAATCAAACGAGCTCATTACAAC) were prepared from oligonucleotides, either PAGE puriﬁed for MS or OligoGold standard for EMSA, purchased from Eurogentec. A ﬂuorescence-based EMSA Kit (Invitrogen) was used according to the manufacturer’s instructions (37) to analyze the binding of CrgA to DNA. EMSA reactions of 10 ml were made up in 150 mM KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris, pH 7.4. The amount of DNA in each reaction was kept constant at 2.5 pmol for experiments with CrgA or 1.5 pmols for experiments with the R55Q CrgA protein. The molarity of CrgA, calculated for an octameric species was varied up to a 4 molar excess over the amount of DNA. After the addition of 2 ml of 6 EMSA-loading