The 70S Ribosome
Phone: +49 - 89 - 8578 2032
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For its abundance (more than 40.000 copies in a E. coli cell), and crucial function as molecular machine responsible for protein synthesis, the 70S ribosome is a relevant target to be analyzed by Cryo-electron Tomography in the framework of visual proteomics pursued within our Department.
We have applied pattern recognition techniques (template matching and scaling index segmentation) for the detection and identification of 70S ribosomes in frozen-hydrated, intact thin cells (Spiroplasma melliferum). Fundamental questions about cellular distribution of ribosomes and spatial interactions between them and with other cell component, e.g. the membrane, can be addressed with our approach.
One of our goals is to gain detailed structural information about this molecular complex in its cellular enviroment. Due to the low signal-to-noise ratio of individual tomograms it is indispensable to average many correctly identified ribosomes in the cytoplasm. This approach has been succesfully applied for Spiroplasma, and allowed us to derived a ribosome density map of moderate resolution (~4-5 nm).
The prokaryotic ribosome has been a subject of structural study for more than five decades. There is available a collection of crystal structures and cryo-electron density maps of high resolution solved by Single Particle Analysis (SPA), which present the ribosome in different conformations and attached to diverse macromolecules. However these approaches generally require isolated material or quite homogeneous in vitro reconstituted systems, which demands a good understanding of the biochemical processes underlying the formation of such complexes. Moreover, transient and flexible interactions are difficult to access by X-ray crystallography or SPA.
As described above, we have developed a strategy based on Cryo-Electron Tomography, template matching, 3D-alignment, 3D-averaging, and 3D-classification, that allows purification in silico of diverse species of 70S ribosome complexes. Detection of densities attached to the ribosome at distinctive positions is possible even in highly heterogenous samples, like in vitro translation systems, or enriched ribosomal fractions. We are applying these methods for a description of topologies of ribosomal clusters and association with other complexes, eg. chaperones.
The image on the right shows an isosurface representation of the 70S ribosome (50S blue; 30S yellow), derived from a crystal structure of E. coli ribosome - Schuwirth et al (2005). The structure is embebed in a sphere of radius ~25 nm. A systematical structural description of unknown ribosome binding macromolecules inside this sphere is one of our goals.
We have integrated the use of molecular visualization software, e.g. Chimera (UCSF; San Francisco) and Amira (Mercury Computer Systems, and Zuse Institute, Berlin), with the application of scripting tools of commercial software for modelling and animations (e.g. 3ds Max, Autodesk), for a visual representation of our experimental results and formulation of hypotheses. Even though Cryo-Electron Tomography basically provides snapshots of cell processes, our experimentally determined spatial arrangments of complexes in conjuncion with known crystal structures could provide a platform for molecular simulations. In an effort to add an adequate physicochemical behavior of macromolecules to our static views of the cell and its complexes, we have started collaboration projects in the field of molecular dynamics.
Dr. Julio Ortiz
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