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Abstract

Subject of these studies was the strong binding complex of actin and myosin V. How the molecular motor protein myosin powers muscles by generating mechanical force out of chemical energy at high efficiency fascinates researchers already for a long time. Myosin V offers the unique possibility to study not only the rigor state but also its preceding strong binding ADP state, which is only transiently present in other myosins. Since the actomyosin complex could not be studied by protein crystallography so far, transmission electron microscopy (TEM) of vitrified protein complexes is the method of choice. New reconstruction strategies were applied, to enhance the interpretable resolution in 3D electron microscopy, together with subsequent molecular dynamics (MD) simulations of protein crystal structures. Such a combined approach allows for almost the same resolution as X-ray diffraction facilitates. As a new – and in principle superior – reconstruction scheme a filtered least squares re- construction algorithm was applied in order to obtain a better defined density localization. Thorough analysis combined with electron tomography studies revealed flexible specimen properties that limit the applicability of the current implementation of the least squares method due to necessary ad hoc assumptions. As a first step to reduce these assumptions iterative helical reconstruction was applied and yielded densities with reliable 8 Å resolution. Following MD simulations were able to resolve functional differences of myosin between the rigor and the strong binding ADP state.