The three-dimensional structure of α-actinin obtained by cryoelectron microscopy suggests a model for Ca2+-dependent actin binding

The three-dimensional structure of α-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. α-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 ° to 60 °. Interpretation of the 15 Å-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of α-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca2+-dependent actin binding of non-muscle isoforms.