Domain flexibility in the 1.75 Å resolution structure of Pb²⁺-calmodulin

Calmodulin (CaM) regulates a variety of cellular processes by interacting with a large number of proteins in a Ca²⁺-dependent manner. Conformational flexibility plays a key role in CaM function, although the full extent and detailed features of this flexibility are not fully characterized. Here, the 1.75 Å resolution crystal structure of Pb²⁺-bound Paramecium tetraurelia CaM crystallized in a previously unobserved monoclinic lattice is reported. Pb²⁺-CaM is disordered in this new lattice and only a portion of each of the two molecules in the asymmetric unit can be modeled. Comparison of the structures of Ca²⁺-CaM and Pb²⁺-CaM show close agreement in the C-terminal domain but significant structural differences in the N-terminal domain. In addition, translation–libration–screw (TLS) refinement and Rosenfield difference analysis reveal inter-helical flexibility in the metal-bound N-terminal domain of the protein that is absent in the metal-bound C-­terminal domain and indicates that the two structurally similar domains of CaM are dynamically distinct. These results demonstrate that TLS refinement and Rosenfield difference analysis allow detailed information about macromolecular flexibility to be extracted from X-ray diffraction data even when the crystal lattice prohibits full manifestation of this flexibility.