sedimentation coeﬃcient and Stokes radius. The ﬁrst 30 interference scans were analyzed for both samples, allow- ing for frictional ratios up to 2.5. A resolution of 50 was used for the calculations. Sedimentation coeﬃcients were computed for atomic models using the program HYDROPRO (43). RESULTS Overall structure of CrgA We ﬁrst solved the structure of the regulatory domain of the protein (residues 89–300) using selenomethionine-sub- stituted protein by multiple wavelength anomalous disper- sion to 2.3 A˚ resolution. This model was then used to determine the structures of the full-length protein in both orthorhombic and monoclinic crystal forms by molecular replacement to resolutions of 3.2 A˚ and 3.8 A˚ , respectively. As expected, each CrgA subunit of the full- length protein comprises an N-terminal DBD, connected via a long LH to a C-terminal regulatory domain (Figure 1). The DBD contains a wHTH motif (a1, a2, a3, b1 and b2) that is comparable to that of other wHTH superfamily members superimposing with a root mean-square deviation (RMSD) of 2.3–3.0 A˚ (for 53–58 common Ca residues) (Supplementary Figure S1). As expected the position of the a2–a3 (residues 21–41) is extremely well conserved while the position and length of the wing vary within the family. The regulatory domain contains two subdomains RD-I (residues 89–162 and 265–299) and RD-II (residues 163–264). RD-I con- sists of a core of four parallel and one anti-parallel b strands (b3, b4, b5, b6 and b13) sandwiched between a helices (a5–a6, a7 and a11) and a 310 helix (Z2). The b6 and b13 strands cross over between the two subdomains. RD-II contains three a helices (a8, a9 and a10), a 310 helix (Z1) and a series of b strands (b7–b12) (Figure 1). The putative eﬀector pocket, located at the interface of RD-I and RD-II, is large with a solvent-accessible surface area of 603 A˚ 2 and a volume of 1072 A˚ 3 (35). The residues lining the pocket are predominantly non-polar (66%) including four phenylalanine residues (residues F164, F193, F242 and F268) suggesting that the potential eﬀec- tor is largely hydrophobic in nature. The only clue as to the identity of the eﬀector, which we presume modulates the activity of CrgA, is the observation that treatment of Neisseria with a-methylene–g-butyrolactone (MBL) has been shown to induce CrgA-dependent expression of mdaB (18). However in E. coli MBL is not a direct sub- strate of the MdaB enzyme and in a thermal shift (ThermoFluor) assay, that can be used to assess ligand binding (44), we observed no evidence of a direct interac- tion between MBL and CrgA suggesting that MBL is acting indirectly (data not shown). Novel octameric assembly of CrgA Although the fold of each CrgA subunit is similar to that observed for CbnR, the overall assembly of the complex is strikingly diﬀerent. CbnR is tetrameric whereas CrgA assembles into square-like hollow octameric rings with dimensions of 125 125 67 A˚. The regulatory domains Nucleic Acids Research, 2009, Vol. 37, No. 14 4549 Figure 1. CrgA fold and domain architecture. are sandwiched between the DBDs that are located in pairs (DBD pairs) at the four corners of the disc. Viewed from above, the hollow of the octamer has a cross-like shape with a distance of 95 A˚ between oppos- ing LHs and 50 A˚ between opposing regulatory domains (Figure 2). On ﬁrst inspection the octamer, which occupies one asymmetric unit, appears to have 4-fold symmetry. However, there are signiﬁcant diﬀerences in the orienta- tion of the LHs and DBDs within the eight subunits, which break this apparent symmetry. Due to the nature of the assembly, each CrgA subunit contacts just two of the other subunits. Each subunit forms an interface with one adjacent subunit, principally through their equivalent N-terminal domains, (residues 1–89, N-term interface) and to a second subunit, through their regulatory domains (RD interface) (Figure 2). The sizes of these two interfaces are similar, each burying an average surface area of 1300 A˚2. The RD interface contains two sets of equivalent inter- actions, between residues of the RD-I and RD-II on the adjacent chain while the N-terminal interface is primarily formed by dimerization of the LHs through intermolecu- lar anti-parallel coiled coil interactions (Figures 4A and 6D). The question arises as to whether the octameric assem- bly of CrgA observed in the crystal structure is relevant to the protein in solution and hence the biologically active form of the protein. Therefore, the protein was analyzed by non-denaturing nano-electrospray mass spectrometry (MS) to determine the precise oligomeric state of CrgA in solution,. The results showed an intense and well-resolved charge state series, from +36 to +43, in the 6500–8500 m/z range, which corresponded to an octa- meric assembly for CrgA (Figure 3A). The insert in Figure 3A conﬁrms the presence of one dominant