Ca2+-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy

Muscle contraction is regulated by the intracellular Ca2+ concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca2+-sensitive switch that controls contraction. Ca2+ binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca2+ have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to ∼35 Å resolution. With Ca2+, the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca2+, the main body of troponin was shifted by ∼30 Å towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by ∼35 Å supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than ∼12 Å. Therefore tropomyosin shifted differentially without Ca2+. With Ca2+, tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.