Single particle analysis of relaxed and activated muscle thin filaments

The movement of tropomyosin from actin's outer to its inner domain plays a key role in sterically regulating muscle contraction. This movement, from a low Ca 2+ to a Ca 2+ -induced position has been directly demonstrated by electron microscopy and helical reconstruction. Solution studies, however, suggest that tropomyosin oscillates dynamically between these positions at all Ca 2+ levels, and that it is the position of this equilibrium that is controlled by Ca 2+ . Helical reconstruction reveals only the average position of tropomyosin on the filament, and not information on the local dynamics of tropomyosin in any one Ca 2+ state. We have therefore used single particle analysis to analyze short filament segments to reveal local variations in tropomyosin behavior. Segments of Ca 2+ -free and Ca 2+ -treated thin filaments were sorted by cross-correlation to low and high Ca 2+ models of the thin filament. Most segments from each data set produced reconstructions matching those previously obtained by helical reconstruction, showing low and high Ca 2+ tropomyosin positions for low and high Ca 2+ filaments. However, ∼20% of segments from Ca 2+ -free filaments fitted best to the high Ca 2+ model, yielding a corresponding high Ca 2+ reconstruction. Conversely, ∼20% of segments from Ca 2+ -treated filaments fitted best to the low Ca 2+ model and produced a low Ca 2+ reconstruction. Hence, tropomyosin position on actin is not fixed in either Ca 2+ state. These findings provide direct structural evidence for the equilibration of tropomyosin position in both high and low Ca 2+ states, and for the concept that Ca 2+ controls the position of this equilibrium. This flexibility in the localization of tropomyosin may provide a means of sterically regulating contraction at low energy cost.