High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures.

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.

Hypothesis on radar sensing and communication by hornets: comments on their antennal organulles and electrical activity.

Our study shows that the antenna of the hornet is densely covered from base to tip with six types of organulles, namely, a trichoid organulle, about 23 microm in length, a campaniform organulle which is 12 microm long, a heretofore undescribed structure measuring about 9 microm in length which we have now named the Agmon organulle, a rather flattened placoid organulle measuring about 25 microm in length and not projecting on the antennal surface, and finally rounded structures about 3 microm in diameter which are indented in the cuticle and resemble extraretinal photoreceptors. All the above-mentioned structures occur in the workers and drones, and the latter also possess an additional structure, namely, the tyloid which is about 254 microm long. Such organulles as protrude from the antennal surface are mostly orientated distally and their length is about half that of similar organulles on the hornets body. Yet their density on the antenna is greater than elsewhere on the body and in fact on all the antennal segments (12 in the workers and 13 in the drones) the entire surface area seems to be occupied by them. The most numerous are the trichoids, while the other organulles mentioned are fewer in number. In measuring the electric properties of the antennae, we obtained the following values: 10-80 nanoAmpers (nA), 60-100 milliVolt (mV) and several scores of Mega Ohms (Momega) in the dark, as compared to 10-20 nA, 150-200 mV and few Momega under illumination. We found that in many respects, the cuticle in the antenna behaves like an organic semiconductor possessing thermophotovoltaic (TPV) properties. Apart from contemplating that the role of the antennal organulles is as a mechanoreceptor, a chemoreceptor or a combination of the two, we also raise the conjecture that these organulles might serve as elements that pick up and broadcast at submillimetric wavelengths, and that the electric energy extant in the vespan antennae supplies the energy source for this kind of activity.

Structure of functionally activated small ribosomal subunit at 3.3 angstroms 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 particle's 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.

Targeting exposed RNA regions in crystals of the small ribosomal subunits at medium resolution.

Within the framework of ribosomal crystallography, the small subunits are being analyzed, using crystals diffracting to 3 A resolution. The medium resolution electron density map of this subunit, obtained by multiple isomorphous replacement, show recognizable morphologies, strikingly similar to the functional active conformer of the small ribosomal subunit. It contains elongated dense features, traceable as RNA chains as well as globular regions into which the structures determined for isolated ribosomal proteins, or other known structural motifs were fitted. To facilitate unbiased map interpretation, metal clusters are being covalently attached either to the surface of the subunits or to DNA oligomers complementary to exposed ribosomal RNA. Two surface cysteines and the 3' end of the 16S ribosomal RNA have been localized. Targeting several additional RNA regions shed light on their relative exposure and confirmed previous studies concerning their functional relevance.

The small ribosomal subunit from Thermus thermophilus at 4.5 A resolution: pattern fittings and the identification of a functional site.

The electron density map of the small ribosomal subunit from Thermus thermophilus, constructed at 4.5 A resolution, shows the recognizable morphology of this particle, as well as structural features that were interpreted as ribosomal RNA and proteins. Unbiased assignments, carried out by quantitative covalent binding of heavy atom compounds at predetermined sites, led to the localization of the surface of the ribosomal protein S13 at a position compatible with previous assignments, whereas the surface of S11 was localized at a distance of about twice its diameter from the site suggested for its center by neutron scattering. Proteins S5 and S7, whose structures have been determined crystallographically, were visually placed in the map with no alterations in their conformations. Regions suitable to host the fold of protein S15 were detected in several positions, all at a significant distance from the location of this protein in the neutron scattering map. Targeting the 16S RNA region, where mRNA docks to allow the formation of the initiation complex by a mercurated mRNA analog, led to the characterization of its vicinity.

Metal compounds as tools for the construction and the interpretation of medium-resolution maps of ribosomal particles.

Procedures were developed exploiting organometallic clusters and coordination compounds in combination with heavy metal salts for derivatization of ribosomal crystals. These enabled the construction of multiple isomorphous replacement (MIR) and multiple isomorphous replacement combined with anomalous scattering medium-resolution electron density maps for the ribosomal particles that yield the crystals diffracting to the highest resolution, 3 A, of the large subunit from Haloarcula marismortui and the small subunit from Thermus thermophilus. The first steps in the interpretation of the 7. 3-A MIR map of the small subunit were made with the aid of a tetrairidium cluster that was covalently attached to exposed sulfhydryls on the particle's surface prior to crystallization. The positions of these sulfhydryls were localized in difference Fourier maps that were constructed with the MIR phases.

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallograhy.

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.

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.

The suitability of a monofunctional reagent of an undecagold cluster for phasing data collected from the large ribosomal subunits from Bacillus stearothermophilus.

An electron density map of the large ribosomal subunit from Bacillus stearothermophilus was obtained at 26 A resolution by single isomorphous replacement (SIR) from a derivative formed by specific quantitative labeling with a dense undecagold cluster. For derivatization, a monofunctional reagent of this cluster was bound to a sulfhydryl group of a purified ribosomal protein, which was in turn reconstituted with core particles of a mutant lacking this protein. The native, mutated, and derivatized 50S ribosomal subunits crystallize under the same conditions in the same space group. Under favorable conditions, crystals of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different packing. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the noncrystallographic symmetry found by Patterson searches as well as with the motif observed in electron micrographs of thin sections of the crystals. These studies established that phase information can be obtained from heavy metal clusters, even when the crystals under investigation are unstable and weakly diffracting. These results encouraged further effort at the construction of specifically derivatized crystals from other ribosomal particles that diffract to higher resolution.