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Developing Cryo Electron Microscopy Tool for Nanomachines

  • Author(s): Ding, Ke
  • Advisor(s): Zhou, Z. Hong
  • Sun, Ren
  • et al.
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Abstract

Large biomolecular complexes often work as nanomachines to directly process molecules, which requires these complexes to adapt different conformations at different states. With the recent rapid development of cryo electron microscopy (cryoEM) technique, both data quality and quantity have improved. As a result, more heterogeneities in the dataset can be detected. How to turn these observed heterogeneities to structural dynamics at the molecular level is a pressing topic in the field of structural biology. In this dissertation, the strong relation between unnatural air-water interfaces in the cryoEM sample and heterogeneities found in structures is proposed. To demonstrate that imaging nanomachines in situ is a reliable way to understand dynamics, two nanomachines, the vault organelle and reovirus polymerases, are resolved to near-atomic resolution, proving cryoEM is a powerful tool to find heterogeneities existing in the dataset. To further understand the working mechanism of reovirus RNA-dependent RNA polymerase (RdRp), different states are induced in two different reoviruses and the dynamics of reovirus RdRp are revealed from the conformational changes observed in the assembled, functional transcriptional complexes (with RdRp and genome both inside the capsid). These works also demonstrate that the classification focusing on the area of interest is an effective tool to resolve heterogeneities, much like a divide-and-conquer approach. When the nanomachine’s structures are resolved in situ, the detected heterogeneities become informative in understanding the nanomachine’s function, particularly for polymerases.

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This item is under embargo until June 11, 2020.