Structure and Atomic Model of Myosin Filaments from Lethoerus Indicus Using Cryo-Electron Microscopy

Cryo-electron microscopy (or cryoEM) is a rapidly expanding field with many research groups developing new methods and hardware every day. cryoEM is now on its way to replace X-ray crystallography as the method of choice to study structure. The ability to study proteins in their native environment using single-particle reconstruction and cryo-electron tomography is exclusive to cryoEM. Advances in detectors and sample preparation techniques are astonishing. The subject of this dissertation is a perfect example of the kind of work that cryoEM makes possible. Our focus is on understanding the muscle and sarcomeric proteins, and the context is an important part of the knowledge gap. Myosin filaments are several microns long, and crystallography is not an option unless we take tiny segments out of context before studying them. Myosin filaments are too large for single-particle reconstruction, and we can only resolve their structure thanks to their helical symmetry. Having a strictly followed helical symmetry helps the cryoEM study of many filamentous structures. Some recent examples are microtubules, α-synuclein, and decorated actin filaments. Myosin filaments play a central role in muscle contraction, and their atomic model can teach us a lot about muscle structure and function. Myosin heads have been the focus of structural studies, and to this day, we have only resolved the backbone structures in two species (and working on the third one). This dissertation begins with an introduction to structural biology from the point of view of a physics student. Basic ideas in biochemistry that relate to the topic are introduced before discussing cryoEM background from image formation to data analysis. We then go through muscle structure and components and finish the chapter with a brief history of the coiled-coil structure. Chapter 2 is an expanded version of the published atomic model to a myosin II coiled-coil in its native environment. We started from the existing knowledge in protein sequence, added our piece to the puzzle, and tried to provide answers to some unexplained phenomena observed in X-ray studies of muscle fibers. Chapter 3 discusses the complete atomic model for the filament, which we hope to improve and publish soon and share our hypothesis about the dynamics of myosin filaments in muscle contraction and a close look at some disease-causing mutations. This model could be the basis of many new simulations in the muscle field for computationally oriented researchers. For future work, the muscle sarcomere can be a very attractive specimen for newer tomography techniques and yield exceptionally high-resolution results in context because of the order and helical symmetry. There is also some computational work we are doing on assessing the error and uncertainty in helical parameters calculated in our field but they are not discussed in this dissertation.