The Complete Structure of the Mycobacterium smegmatis 70S Ribosome

Hentschel, Jendrik; Burnside, Chloe; Mignot, Ingrid; Leibundgut, Marc; Boehringer, Daniel; Ban, Nenad

doi:10.1016/j.celrep.2017.06.029

Cited by 76 publications

(106 citation statements)

References 77 publications

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“…High-resolution structures are now available for many ribosomes purified from bacteria, including Haloarcula marismortui (the first 50S subunit structure published in 2000), 2 Thermus thermophilus, 3 E. coli, 4 B. subtilis, 5 and Mycobacterium smegmatis. 6 The first eukaryotic ribosome was determined for the yeast, Saccharomyces cerevisiae, in 2010. 7 Since then, cryo-EM structures with 2.5–5Å resolution have been reported for the 80S ribosomes from human, 8 Drosophila , 9 Plasmodium falciparum , 10 and Trypanosoma cruzi , 11 and for the mammalian mitochondrial and spinach chloroplast ribosomes.…”

Section: Introductionmentioning

confidence: 99%

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells

et al. 2017

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Solid-state NMR is a powerful tool for quantifying chemical composition and structure in complex assemblies and even whole cells. We employed N{P} REDOR NMR to obtain atomic-level distance propensities in intact 15N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorous was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen contributions to the REDOR spectra, the strongest dephasing emerged from the dipolar couplings to phosphorous involving nitrogen peaks ascribed primarily to rRNA and the weakest dephasing arose from protein amide nitrogens. This approach is applicable to any macromolecular system and provides quantitative comparisons of distance proximities between shift-resolved nuclei of one type and heteronuclear dephasing spins. Enhanced molecular specificity could be achieved through the use of spectroscopic filters or specific labeling. Furthermore, ribosome 13C and 15N CPMAS spectra were compared with those of whole cells from which the ribosomes were isolated. Whole-cell signatures of ribosomes were identified and should be of value in comparing overall cellular ribosome content in whole-cell samples.

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Section: Introductionmentioning

confidence: 99%

Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells

et al. 2017

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“…The Ms bL25 has two functional domains, the NTD interacts with the 5S rRNA, and the CTD is sandwiched between the uL16 (green) and H38 of 23S rRNA. also been reported in the recent mycobacterial ribosome structure [147] ( Figure 3.22). The density observed for these two helices is weak in our map and thus the filtered maps were used to build the models in this region.…”

Section: Figure 321 Ms Bl25 With Its Two Functional Domainssupporting

confidence: 80%

“…The density for bS21 was also not observed in the Ms and Mtb ribosome structures published recently [146][147][148]. bS21 is a late ribosome binding protein.…”

Section: Figure 315 R-protein Us16 Interaction With Us4 In Ms 30s (mentioning

confidence: 71%

“…The density observed for these two helices is weak in our map and thus the filtered maps were used to build the models in this region. This insertion is conserved across Mycobacteria and is 4 nt longer in Mtb [147]. H54a: H54a is a Mycobacteria-specific 113 nt long insertion in H54 at position G1532 which emerges at the solvent side of LSU near the L1 stalk, in the domain III of the 23S rRNA (Figure 3.25).…”

Section: Figure 321 Ms Bl25 With Its Two Functional Domainsmentioning

confidence: 99%

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Cryo-EM structure of the Mycobacterium smegmatis ribosome

Mishra¹

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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

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“…Mycobacteria, for example, have very distinct translational features compared with the model E. coli system (39, 48, 49). Recent structural information about the mycobacterial ribosome identified new ribosomal protein subunits, but these new subunits, bL37 and bS22 were not present in all ribosomes identified by cryo-electron microscopy (34, 50, 51).…”

Section: Discussionmentioning

confidence: 98%