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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

Wulf, Sarah Elise Freyja

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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.

Item Type: Dissertation
Supervisor: Schröder, Prof. Dr. Rasmus R.
Date of thesis defense: 16 April 2014
Date Deposited: 09 May 2014 07:03
Date: 2014
Faculties / Institutes: The Faculty of Physics and Astronomy > Dekanat der Fakultät für Physik und Astronomie
Subjects: 530 Physics
Controlled Keywords: Transmissionselektronenmikroskopie
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