Visualizing supramolecular organizations of 70S ribosomes in vitro and inside the cell
We use electron cryo-tomography (ECT) in conjunction with advanced image analysis techniques to analyze relevant associations between macromolecular complexes. The cryo-fixation of samples allows to gain structural information in a close-to-native state, while the application of electron tomography results in three-dimensional (3D) models of the objects under scrutiny. Our techniques are suitable for the analysis of samples with a variable degree of complexity, from isolated molecular machines to whole cells, including mixtures of isolated components. Therefore, we can describe possible interactions between cellular machineries under the controlled conditions of an in vitro assay and also provide direct evidence of such associations inside the cell.
Using these methods, we have been able to describe the 3D organization of densely packed polysomes formed in an in vitro translation system supplemented with truncated mRNAs (Brandt, et. al, 2009, Cell 136:261-271). We also analyzed the 3D structure of a complex formed by the specific association of two 70S ribosomes, commonly referred as the 100S ribosome or hibernating ribosome. The structure of the 100S ribosome, which we originally solved in ribosome enriched fraction from starved E. coli cells, was clearly detected also in intact E. coli cells grown under nutritional stress. This lays to rest speculations that ribosomal dimers may be an artefact caused by the buffer conditions used for purification. This was the first time that a novel 3D structure was derived in situ imaging the crowded bacterial cytoplasm (Ortiz, et al. 2010 JCB 190:613-620).
Methodological challenges, new perspectives
Our ongoing efforts aim to improve the performance of all our techniques and to achieve better cellular tomography by incorporating new technologies. Focused Ion Beam (FIB) milling, a method for physical sectioning of vitrified cells, is a recent development in sample preparation that is helping us to apply ECT to originally thick specimens (>500 nm). Dual-axis tilt tomography has been included in our pipeline to gain a more isotropic resolution. The use of a Direct Electron Detector coupled to energy-filtering in a cutting-edge TEM is offering us a significant improvement in the spatial resolution of tomograms. We expect that these instrumental novelties and further developments in algorithms for image processing will allow us to address fundamental questions about cellular distribution of ribosomes and other cell component in more demanding samples (i.e., fast growing E. coli cells).
Roberts E., Magis A., Ortiz J.O., Baumeister W., Luthey-Schulte, Z.: Noise contributions in an inducible genetic switch: a whole-cell simulation study. PLoS Computational Biology, 7:e1002010, 2011
Ortiz J.O., Brandt F., Matias V.R.F., Sennels L., Rappsilber J., Scheres S.H.W., Eibauer M., Hartl F.U. and Baumeister W.: Structure of Hibernating Ribosomes Studied by Cryoelectron Tomography in vitro and in situ. J. Cell. Biol., 190:613-621, 2010
Brandt F., Etchells S. A., Ortiz J. O., Elcock A. H., Hartl F. U. and Baumeister, W.: The Native 3D Organization of Bacterial Polysomes. Cell, 136:261-271, 2009
Ortiz J.O., Förster F., Kürner J., Linaroudis A.A. and Baumeister W.: Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition. J Struct Biol, 156:p. 334-341, 2006