RIBOSOME 70S

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

Mapping of 70S Ribosomes in intact cells

For its abundance (6.000 to 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 Cryoelectron Tomography (CET) in the framework of visual proteomics pursued within our Department. 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. 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 and starved Escherichia coli).

To recover translation information from noisy tomograms, it is indispensable to average many copies of ribosomes in similar state. 3D-averaging and 3D-classification of subtomograms containing identified ribosomes allowed us to derive ribosome density maps of moderate resolution for Spiroplasma and starved E. coli. A first example of ribosomal supramolecular organization detected in E. coli is the dimerized form of 70S ribosomes associated with starvation (100S or hibernating ribosomes). These dimers had been previously observed only in isolated fractions. With increasing improvements in subtomograms classification methods and tomograms quality, we expect to identify in situ ribosomes associated to smaller complexes. Native 3D-organization of ribosomal clusters double-2

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 demand 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,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 developed. We applied these methods for a description of 3D-organizations of ribosomal clusters. We showed that E. coli ribosomes adopt two preferential relative orientations in densely-packed polysomes formed in vitro. These alternative manners of ribosomal pairing result in variable 3D polysomal organizations, i.e, pseudo-planar or pseudo-helical polysomes (See movie). More recently, we also proved that it was possible to purify in silico a particular ribosomal arrangement of the two 70S ribosomes from heterogeneous cytosolic fraction. That is the case of the above mentioned hibernating ribosomes, reversibly inhibited ribosomal dimers which show additional associated densities close to the exit of the mRNA tunnel. Molecular environment of 70S Ribosome 70S_enviroment_medium

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. Visualization of cellular complexity

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.