4550 Nucleic Acids Research, 2009, Vol. 37, No. 14 Figure 2. Octameric assembly of CrgA found in the full-length structure. oligomeric state over a large m/z range. Additionally, a small amount of hexameric CrgA was detected by MS, (hexamer peaks are identiﬁed by black circles). The mea- sured mass of the octamer was 273 542 25 Da, which is slightly greater than the predicted mass of 272 919 Da. This discrepancy in mass is explained by the inability to achieve complete species desolvation of the complex with the ‘soft’ ionization methods required to maintain subunit interactions. This results in the retention of water and buﬀer molecules within the protein complex ions that are detected (45). The octameric stoichiometry was con- ﬁrmed by activating the complex using collision-induced dissociation (CID) (46). This results in a proportion of the octamer dissociating into a heptamer as a monomer is unfolded and ejected from the intact assembly carrying with it a disproportionate amount of the overall charge of the complex (Figure 3B insert). Due to the greater acti- vation of the complex during CID experiments, enhanced peak resolution is achieved, enabling a more accurate mass measurement and conﬁrming the octameric assembly of CrgA. As described below, the results of AUC experi- ments also conﬁrmed the octameric state of the CrgA protein in solution (Figure 5A). Comparison of the assembly of CrgA and CbnR The most notable diﬀerence between CrgA and CbnR is the orientation of their DBD and LH. In all eight subunits of CrgA the LH folds back toward the regulatory domain whereas in CbnR in the compact form (chains A and P), the LH folds back toward the regulatory domain. While in the extended form (chains B and Q), the LH extends away from the regulatory domain with a rotation of 808 (Figure 4). The hinge region of LTTRs, ﬁrst identiﬁed in CbnR, largely controls the orientation of the DBD and LH. Cross-comparison (Supplementary Figure S2) of the eight chains of CrgA indicates that there is a signiﬁcant amount of rotational freedom of the LH around the hinge but that there is no large pivot movement of the regulatory domain away from the DBD–LH as observed between the compact and extended forms of CbnR (2). In CrgA, the hinge (H88, E89, I90 P91, Q92 and G93) contains a PXG motif whereas in CbnR, there are two glycine residues (GXXG) in this region. The more constrained geometry of a proline compared to a glycine residue in the hinge of CrgA may cause it to have less structural freedom than CbnR, which can adopt two markedly diﬀerent conforma- tions. The fact that the PXG motif is conserved within close homologs, though not within the LTTR family as a whole, suggests that it may be functionally important. Signiﬁcantly, in CbnR there is a third type of interface formed between the two aV helices (residues S201 to H212) of RD-II in chains A and P (both in the compact form), which stabilizes the central core of the tetramer (2). In CrgA, this helix is replaced by a b strand (b8)