Structure of the thin filament in native skeletal muscles reveals its interaction with nebulin and two distinct conformations of myosin

Abstract

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament "molecular ruler" and the impact of nemaline myopathies mutations that will aid future development of therapies.