@phdthesis{Song2021,
  author    = {Song, Boyuan},
  title     = {Structural and functional studies of \(Saccharomyces\) \(cerevisiae\) Ccr4-Not complex with Electron microscopy},
  doi       = {10.25972/OPUS-21652},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216527},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The degradation of poly-adenosine tails of messenger RNAs (mRNAs) in the eukaryotic cells is a determining step in controlling the level of gene expression. The highly conserved Ccr4-Not complex was identified as the major deadenylation complex in all eukaryotic organisms. Plenty of biochemical studies have shown that this complex is also involved in many aspects of the mRNA metabolism, but we are still lacking the detailed structural information about its overall architecture and conformational states that could help to elucidate its multifunction and the way it is coordinated in the cells. Such information can also provide a basis to finding a possible way of intervention since the complex is also involved in some diseases such as cancer and cardiovascular disorders in humans. Meanwhile, the single particle Cryo-EM method has been through a "resolution revolution" recently due to the use of the newly developed direct electron detectors and has since resolved the high-resolution structures of many macromolecular protein complexes in their near-native state. Therefore, it was employed as a suitable method for studying the Ccr4-Not complex here. In this work, the Falcon 3EC direct detector mounted on the 300kV Titan Krios G3i Cryo-EM was evaluated for its practical performance at obtaining high-quality Cryo-EM data from protein samples of different molecular sizes. This served as a proof of principle for this detector's capabilities and as a data collection guidance for studying the macromolecular complexes, such as the Ccr4-Not, when using an advanced high-performance microscope system. Next, the endogenous yeast Ccr4-Not complex was also purified via the immunoaffinity purification method and evaluated using negative staining EM to assess the conditions of the complex before proceeding to sample preparation for Cryo-EM. This has shown that the complex had an unexpected inherently dynamic property in vitro and extra optimisation procedures were needed to stabilise the complex during the purification and sample preparation. In addition, by using the label-free quantitative Mass spectrometry to examine the coimmunoprecipitated complex via different tagged subunits, it was deduced that two of the subunits (Not3/Not5) that shared some sequence similarity might compete for association with the scaffold subunit of the complex. An uncharacterised protein was also identified coimmunoprecipitating with the Caf130 subunit of the yeast complex. Cryo-EM data from the purified complex provided a low-resolution map that represents a surprisingly smaller partial complex as compared to 3D structures from previous studies, although gel electrophoresis and Mass spectrometry data have identified all of the nine subunits of the Ccr4-Not core complex in the sample. It was concluded that due to the presence of many predicted unstructured regions VI in the subunits and their dynamic composition in solution, the native complex could have been spontaneously denatured at the air/water interface during the sample preparation thus limiting the resolution of the Cryo-EM reconstruction. The purified complex was also examined for its deadenylase and ubiquitin ligase activity by in vitro assays. It was shown that the native complex has a different rate of activity and possibly also a different mode of action compared to the recombinant complexes from other species under similar reaction conditions. The Not4 E3 ligase was also shown to be active in the complex and was likely auto-ubiquitinated in the absence of a substrate. Both types of assays have also shown that the conformational flexibility does not seem to affect the enzymatic reactions when using a chemically crosslinked form of the complex for the assay, which implies that there can be other underlying mechanisms coordinating its structural and functional relationship. The findings from this work have therefore moved our understanding of the Ccr4-Not complex forward by looking at the different structural and functional behaviours of the endogenous complex, especially highlighting the obstacles in sample preparation for the native complex in high-resolution Cryo-EM. This would serve as foundation for future studies on the mechanism of this complex's catalytic functions and also for optimising the Cryo-EM sample to generate better data that could eventually resolve the structure to a high-resolution.},
  subject      = {CCR4},
  language  = {en}
}