Cryo-EM structure of the Mycobacterium smegmatis ribosome

Abstract

vi 1.8.1 Mechanism of formation of hibernating ribosome dimers (100S ribosome) 1.8.2 Physiological significance of hibernating ribosome 1.9 Trans-translating Ribosome 1.9.1 Structure of tmRNA 1.9.2 The mechanism of trans-translation 1.9.3 Physiological significance of trans-translation 1.10 Mycobacterial Ribosome 1.11 Cryo-EM 1.12 Aim of this dissertation 2 Materials and Methods 2.1 Materials 2.1.1 Buffers, Media and Salts 2.1.2 Microbiology strains 2.1.3 Buffer Composition 2.1.4 Instruments and accessories 2.1.5 Computer tools and softwares 2.2 Methods 2.2.1 Purification of 70S ribosomes from M. smegmatis 2.2.2 Ribosome protein identification by SDS PAGE and Mass Spectrometry 2.2.3 Purification of 70S ribosomes from M. smegmatis growing in different growth phases 2.2.4 Cryo-Electron Microscopy 2.2.5 Cryo-EM Single Particle Analysis (Data Processing) 2.2.6 Current capabilities and limitations of cryo-EM and Single Particle Analysis 2.2.7 Cryo-EM imaging of M. smegmatis ribosome 2.2.8 Model building and refinement 2.2.9 Model validation 2.2.10 Figure generation vii 3 Results Cryo-EM structures of M. smegmatis ribosomes in different functional states 3.1 Purification profile of M. smegmatis ribosomes in log and stationary phase 3.2 Micrograph inspection 3.3 Image Processing 3.3.1 2D classification 3.3.2 3D classification and 3D refinement 3.3.3 Resolution estimation and Resmap calculation for the 70S P/P state maps 3.3.4 Model Validation 3.3.5 Resolution estimation for the Hibernating and Trans-translating maps 3.4 Structure of M. smegmatis 70S ribosome in P/P state 3.4.1 M. smegmatis small ribosomal subunit 3.4.2 M. smegmatis large ribosomal subunit 3.4.3 Species specific alteration in the 23S rRNA of the LSU in the M. smegmatis ribosome 3.4.4 Species specific alteration in r-proteins clustered near the functional sites of M. smegmatis ribosome 3.5 Structure of M. smegmatis 70S ribosome in the Hibernating state 3.5.1 Hibernation Promoting Factor 3.5.2 Structure of mRNA entry and exit sites of M. smegmatis 70S ribosome 3.6 Trans-Translating state of M. smegmatis 70S ribosome 3.6.1 Unique mechanism of tmRNA during trans-translation 4 Discussion 5 Conclusion 6 References 7 Appendix viii 8 Online access of cryo-EM maps and models 162 9 Author's publications related to this thesis during the