Reference Free Single Particle Analysis Of Reconstituted Thin Filaments

Abstract

A detailed three-dimensional structure of the muscle thin filament is required in order to understand its regulation. To this end we have applied a reference free single particle analysis approach to electron microscope images of negatively stained reconstituted thin filaments from skeletal actin and cardiac tropomyosin and troponin. The filaments were prepared in a low Ca2+ buffer. For image analysis the filaments were segmented into ∼800A long particles centred on the troponin complex. Density attributable to troponin and tropomyosin is readily identifiable in the two-dimensional class averages and the three-dimensional reconstruction. The data have previously been analysed using a model-based single particle method (Pirani et al., 2005, 2006). Our non-model based approach and novel strand averaging procedure has enabled us to quantify directly the stagger or axial rise between adjacent troponin complexes (∼27.7A). Comparison with our previous analysis of native thin filaments indicates that reconstituted filaments assemble with the same arrangement of troponin as in vivo, viz .in register on both helical strands with a ∼40 nm repeat. This indicates that troponin and tropomyosin can organise themselves on actin filaments without requiring any other sarcomeric proteins.Pirani A., Vinogradova M.V., Curmi P.M., King W.A., Fletterick R.J., Craig R., Tobacman L.S., Xu C., Hatch V., Lehman W. 2006. An atomic model of the thin filament in the relaxed and Ca2+-activated States. J Mol Biol 357(3):707-17.Pirani A., Xu C., Hatch V., Craig R., Tobacman L.S., Lehman W. 2005. Single particle analysis of relaxed and activated muscle thin filaments. J Mol Biol 346(3):761-72.