Biomol PhD Award:
Structural insights into sarcomere organisation by cryo-electron tomography
Zhexin Wang1, Michael Grange1 2, Sabrina Pospich1, Thorsten Wagner1, Ay Lin Kho3, Mathias Gautel3, Stefan Raunser1
1Max Planck Institute of Molecular Physiology, Dortmund, Germany, 2Rosalind Franklin Institute, United Kingdom, 3King's College London Guy's Campus, London, United Kingdom
Sarcomeres are force-generating and load bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and diseases. Here, we determine the molecular architecture of native skeletal sarcomeres and structures of sarcomeric proteins using cryo-focused-ion-beam milling (cryo-FIB) and electron cryo-tomography (cryo-ET). Our three-dimensional reconstruction of the sarcomere reveals molecular details in the A-band, I-band and Z-disc and demonstrates the organisation of the thin and thick filaments and their cross-links. Various in situ structures determined within sarcomeres with sub-tomogram averaging reveal unexpected interactions between actin, myosin, tropomyosin, troponin and α-actinin. The native structure of the previously “nebulous” protein, nebulin, determined at 4.5 Å demonstrates the molecular mechanism underlying its role in stabilising thin filament, in regulating myosin binding and as a “molecular ruler”. The structure establishes the molecular basis for the pathogenicity of nemaline myopathies mutations. In addition, myosin in the rigor-state sarcomere exhibits unique “double-head” and “split head” arrangements. The different conformations of the myosin neck domain, both within one and among different “double-head”, highlight the inherent structural variability of myosin in muscle. Our structures of both the entire sarcomere and individual sarcomeric proteins after cryo-FIB and cryo-ET represent an expansion in capability for in situ structural biology and serve as a foundation for future investigations of muscle diseases.
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