The E41K mutation in TPM2 gene encoding muscle regulatory protein beta-tropomyosin is associated with nemaline myopathy and cap disease. The mutation results in a reduced Ca ²⁺-sensitivity of the thin filaments and in muscle weakness. To elucidate the structural basis of the reduced Ca ²⁺-sensitivity of the thin filaments, we studied multistep changes in spatial arrangement of tropomyosin (Tpm), actin and myosin heads during the ATPase cycle in reconstituted fibers, using the polarized fluorescence microscopy. The E41K mutation inhibits troponin's ability to shift Tpm to the closed position at high Ca ²⁺, thus restraining the transition of the thin filaments from the “off” to the “on” state. The mutation also inhibits the ability of S1 to shift Tpm to the open position, decreases the amount of the myosin heads bound strongly to actin at high Ca ²⁺, but increases the number of such heads at low Ca ²⁺. These changes may contribute to the low Ca ²⁺-sensitivity and muscle weakness. As the mutation has no effect on troponin's ability to switch actin monomers on at high Ca ²⁺ and inhibits their switching off at low Ca ²⁺, the use of reagents that increase the Ca ²⁺-sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.