%0 Generic
%A Wulf, Sarah Elise Freyja
%D 2014
%F heidok:16828
%R 10.11588/heidok.00016828
%T Application of new reconstruction strategies to enhance the interpretable resolution in 3D Electron Microscopy - Studies of Rigor and ADP binding complexes of Actin and Myosin V  
%U https://archiv.ub.uni-heidelberg.de/volltextserver/16828/
%X 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.