Single Particle Analysis: A new approach to solving the 3D structure of myosin filaments

Knowledge of the structure of muscle myosin filaments is essential for a proper understanding of sarcomere structure and how myosin heads interact with the actin filaments to produce force and movement. Two principal methods have been used to define the myosin head arrays in filaments in the relaxed state, namely modelling from low-angle X-ray diffraction data and image processing of electron micrographs of isolated filaments. Analysis of filament images by 3D helical reconstruction, which imposes total helical symmetry on the structure, is very effective in some cases. but it relies on the existence of very highly ordered preparations of straight filaments. Resolutions achieved to date are about 70 &ANGS;. Modelling of X-ray diffraction data recorded from whole relaxed fish or insect Muscles has also been used as an independent method. Although the resolution of the diffraction data is often also about 70 &ANGS;. the effective resolution of the modelling is very much higher than this because additional very high resolution data (e.g. from protein crystallography) is included in the analysis. However, the X-ray diffraction method has to (date provided only limited data on non-myosin thick filament proteins such as C-protein and titin and it cannot provide the polarity of the myosin head arrangement. Both the helical reconstruction and X-ray diffraction techniques have advantages and disadvantages, but their disadvantages are avoided in the new approach of single particle analysis of electron micrograph data. Even using the same micrographs as for helical reconstruction. the resolution can be extended by this method to about 50 &ANGS; or better. In addition, it is not necessary to assume that the myosin filaments are helical, a significant advantage in the case of vertebrate myosin filaments where there is a known crossbridge perturbation. Here we describe the principles of all these approaches, but particularly, that of single particle analysis. We outline the application of single particle analysis to myosin filaments from vertebrate skeletal and insect flight (IFM) muscle myosin filaments.