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Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Å Resolution

The small ribosomal subunit performs the decoding of genetic information during translation. The structure of that from Thermus thermophilus shows that the decoding center, which positions mRNA and three tRNAs, is constructed entirely of RNA. The entrance to the mRNA channel will encircle the message when a latch-like contact closes and contributes to processivity and fidelity. Extended RNA helical elements that run longitudinally through the body transmit structural changes, correlating events at the particles far end with the cycle of mRNA translocation at the decoding region. 96% of the nucleotides were traced and the main fold of all proteins was determined. The latter are either peripheral or appear to serve as linkers. Some may assist the directionality of translocation.

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome–antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

Crystallographic Studies on the Ribosome, a Large Macromolecular Assembly Exhibiting Severe Nonisomorphism, Extreme Beam Sensitivity and No Internal Symmetry

Crystals, diffracting best to around 3 A, have been grown from intact large and small ribosomal subunits. The bright synchrotron radiation necessary for the collection of the higher-resolution X-ray diffraction data introduces significant decay even at cryo temperatures. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small ribosomal subunits, reliable phases have been extracted at medium resolution (5-6 A) and an interpretable five-derivative MIR map has been constructed. For the crystals of the large subunits, however, the situation is more complicated because at higher resolution (2.7-7 A) they suffer from substantial radiation sensitivity, a low level of isomorphism, instability of the longest unit-cell axis and nonisotropic mosaicity. The 8 A MIR map, constructed to gain insight into this unusual system, may provide feasible reasoning for the odd combination of the properties of these crystals as well as hints for future improvement. Parallel efforts, in which electron-microscopy-reconstructed images are being exploited for molecular-replacement studies, are also discussed.

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and x-ray crystallography

BACKGROUND Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography. RESULTS Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown. CONCLUSIONS Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.

The identification of selected components in electron density maps of prokaryotic ribosomes at 7 Å resolution

Crystals of small and large ribosomal subunits from thermophilic and halophilic bacteria, diffracting to 3 A, are being subjected to structural analysis with synchrotron radiation. The bright beam necessary for detecting and collecting the diffraction at the higher-resolution shell causes significant decay even at 25 K. Nevertheless, data collected from native and heavy-atom-derivatized crystals led to the construction of electron density maps of both ribosomal subunits, showing recognizable morphologies and internal features similar to those observed by EM reconstructions of the corresponding ribosomal particle. The main features of these maps include elongated dense regions traceable as well separated RNA duplexes or single strands. Also seen are globular patches of lower density, readily distinguishable from the above, in which folds observed by NMR or crystallography in isolated ribosomal proteins at atomic resolution were detected. The intercomponents contacts identified so far reveal diverse modes of recognition. Metal clusters, attached at selected sites on the particles, are being exploited to facilitate unbiased map interpretation. In this way, two surface proteins were located and several surface RNA strands were targeted.

Ribosomal Crystallography and Heteropolytungstates

Heteropolytungstates play a dual role in ribosomal crystallography. Beside generating phases, one of them, (NH4)6(P2W18O62)14H2O, was found to be extremely useful in inducing post crystallization rearrangements. These led to a significant increase in the internal order of crystals of the small ribosomal subunits from Thermus thermophilus, manifested in a dramatic extension of the resolution of their diffraction patterns, from the initial 7–9 A to 3 A. The current 3.3 A electron density map of this particle, constructed using phases obtained from this W cluster together with other metal compounds, shows the recognizable overall morphology of the small ribosomal subunit. Over 96% of the nucleotides were traced and the fold of all proteins was determined fully or partially. Specific sites were determined independently by covalently bound heavy atom clusters, among them the surface of two proteins and a functional center, the gate for mRNA binding. All tungsten-cluster sites detected in this map are located in close proximity to the proteins of the particle, in positions that may have an influence on the stability and the rigidity of this rather flexible ribosomal subunit.