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In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo–electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament “molecular ruler” and provide a molecular basis for studying nemaline myopathies. Description Clearing up roles of nebulin Thin and thick actomyosin filaments are the key components of muscle. In skeletal muscle, the protein nebulin is essential for the length and strength of the thin filaments, with mutations of nebulin often leading to muscle diseases called nemaline myopathies. Wang et al. used cryo–electron tomography to identify nebulin integrated within the thin filament of native skeletal muscles. The authors determined a near-atomic in situ structure of nebulin and showed how it stabilizes thin filaments and functions as a “molecular ruler. ” The structure of nebulin along thin filaments is key to understanding the pathogenicity of nemaline myopathies. —SMH In situ cryo-EM structures of muscle thin filaments elucidate the structural and regulatory role of nebulin. INTRODUCTION Muscles underpin movement and heart function. Contraction and relaxation of muscles relies on the sliding between two types of filaments—the thin filament [made up of mainly filamentous actin (F-actin), tropomyosin, and troponin] and the thick myosin filament. Additionally, several other proteins are involved in the contraction mechanism, and their mutational malfunction can lead to debilitating and even life-threatening diseases. One such component in skeletal muscle, nebulin, binds to the thin filaments and stabilizes them. It is also responsible for maintaining the length of thin filaments and is involved in regulating myosin binding. Nebulin consists mainly of tandem repeats with different sequences but a conserved SDxxYK motif. Mutations in the nebulin gene are closely linked to a group of muscle diseases called nemaline myopathies. RATIONALE The mechanism underlying nebulin stabilization and the regulation of thin filaments remains nebulous because of missing structural information about the protein. It has been challenging to characterize isolated nebulin because of its enormous size and elongated and flexible nature. To investigate the structure of nebulin in its native environment, we prepared myofibrils from skeletal and cardiac muscle using focused ion beam milling and imaged them using cryo–electron tomography (cryo-ET). With subtomogram averaging, we obtained structures of cardiac and skeletal thin filaments. Because nebulin is only present in skeletal but not cardiac muscle, comparing the thin filament structures allowed us to unambiguously identify and characterize nebulin in the native muscle. RESULTS We resolved nebulin bound to the thin filament within myofibrils isolated from the mouse psoas muscle at near-atomic resolution. In skeletal muscle, two elongated nebulin molecules bind along one actin filament. The structure reveals a 1:1 binding stoichiometry between nebulin repeats and actin subunits. Each nebulin repeat consists of two helices separated by a kink and followed by a loop region. Different nebulin repeats located at different positions along the filament have the same physical length despite their slightly varying sizes, which supports the role of nebulin as a “molecular ruler.” A nebulin repeat interacts with all three neighboring actin subunits though the SDxxYK motif and other conserved charged residues. This explains how nebulin stabilizes the thin filament. Additionally, the position of nebulin on the filament demonstrates that it does not interact with tropomyosin or myosin but likely with a troponin T (TnT) linker. Our reconstruction of myosin shows that the myosin double head exhibits inherent variability within a sarcomere and that nebulin does not alter actin-myosin interactions directly. Therefore, we propose that the myosin-binding regulatory role of nebulin is through its potential interactions with TnT. Nebulin is likely to interact with the TnT linker on two sites, which feature a WLKGIGW motif and a ExxK motif. CONCLUSION Our results show that nebulin is an integral component of the thin filament in skeletal muscle. The interactions between nebulin and other thin filament components set the molecular basis for its functions in thin filament stabilization, length control, and myosin-binding regulation. Our structure of nebulin enables the development of experimental models that further help to reveal how mutations responsible for nemaline myopathies affect nebulin’s function in the sarcomere. The in situ structures of nebulin and myosin illustrated differences from in vitro characterizations and provided structural details relevant in a biology context. Our approach—using focused ion beam milling and cryo-ET to study the proteins of muscles at high resolution—paves the way for studying other muscle components in the future to understand muscle diseases at the molecular level. In situ structure of nebulin on the thin filament from mouse skeletal muscle. Nebulin, resolved at a resolution of 4.5 Å, was identified by comparing cardiac and skeletal thin filament structures. The structure of nebulin reveals the mechanism underlying its function to maintain the length and stability of the thin filament and to regulate muscle contraction. Actin, nebulin, tropomyosin, TnT, myosin heavy chain, myosin essential light chain, and myosin regulatory light chain are colored in green, magenta, light blue, dark blue, yellow, orange, and red, respectively.