We have developed a method to place an EF-lobe in a coordinate system that recognizes the similarity of its two EF-hand domains as well as their relationship by a pseudo-two fold axis, z. The x-axis connects the center of mass, calculated from α-carbons of helices E1 and F1, with the center of mass of E2 and F2. The resulting coordinate system is intrinsic to each EF-lobe and requires no comparison with other EF-lobes. It has provided an intuitive and informative way to compare EF-lobes and especially those changes associated with calcium and/or target binding. We analyzed the EF-lobes of calmodulin and of other subfamilies with four EF-hands. We have rationalized a complex pattern of changes of conformation associated with calcium coordination and effector binding as observed in different subfamilies of EF-hand proteins. Proteins 2012. © 2012 Wiley Periodicals, Inc.