Ribosome Crystals Research Articles

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

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. 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. 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.

On the ab initio solution of the phase problem for macromolecules at very low resolution: the few atoms model method.

A method is proposed for the solution of the phase problem at very low resolution for macromolecules. It generates randomly a very large number of models, each consisting of a few (two to ten) pseudo-atoms. The corresponding amplitudes are used for selecting a subset of 'best' models by choosing those with the highest correlation with experimental values. The phases calculated from these 'best' models are analysed by a clusterization procedure leading to a few possible solutions, from which the correct one can be recognized by simple additional criteria. This method has been successfully applied to the neutron diffraction data of the AspRS-tRNA(Asp) complex at 50 A resolution and to data calculated from a model ribosome crystal at 60 A resolution.

Anomalous Dispersion with Edges in the Soft X-ray Region: First Results of Diffraction from Single Crystals of Ribosomes Near the K-Absorption Edge of Phosphorus

X-ray diffraction from a single crystal of the small ribosomal subunit of Thermus thermophilus was measured at five wavelengths near the K-absorption edge of phosphorus. The intensity of the low-order diffraction peaks varies strongly with the wavelength. It is influenced by the dispersion of contrast due to the P atoms of the ribosomal ribonucleic acid and by that of absorption, in comparable amounts.

Crystallography of halophilic ribosome: The isolation of an internal ribonucleoprotein complex

Crystals of 5OS ribosomal subunits from Haloarcula marismortui diffracting to 2.9 Å resolution were grown. Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation. At ambient temperature, all ribosomal crystals decay upon the first instance of X-irradiation. To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed. Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage. They also can be stored for months, to resume interrupted measurements. To assist the interpretation of the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaL1 and a stretch of H23S rRNA was isolated from the halophilic ribosome. The fragments of the 23S rRNA protected by the protein from nuclease digestion were sequenced. Alignment of the sequences of some archaebacterial L1-specific RNA fragments to the corresponding parts of eubacterial and eukaryotic rDNAs, localized the sequence identities to two distinct regions. Chimeric complexes were reconstituted with the corresponding E. coli ribosomal components, indicating a rather high homology, despite the evolution distance. A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E. coli ribosomal RNA.

Crystallographic, Biochemical and Genetic Studies on Halophilic Ribosomes

Summary Ribosomal particles from Haloarcula marismortui were crystallized. The best crystals, diffracting to 2.9 A resolution and yielding crystallographic data of reasonable quality were grown from the large ribosomal subunits. Attempts at crystallization of functional ribosomal complexes are in progress, benefiting from experience gained from crystals of ribosomes of an extreme thermophilic bacterium, Thermus thermophilus . For obtaining phase information, a monofunctional reagent was prepared from an undecagold cluster, by attaching to it a chemically reactive handle, specific for sulfhydryl moieties. Heavy atom derivatives were prepared by binding this cluster to exposed sulfhydryls prior to the crystallization. Cores of halophilic ribosomal particles, lacking four ribosomal proteins, were prepared using dioxane. All detached proteins could be fully reconstituted. However, blocking the -SH group of one of them (HmaL11), prevented its incorporation into the core particles. The so obtained depleted 50S subunits crystallize under the same conditions as native ones and show apparent isomorphism with them. Most of the genes of several r-proteins were cloned. These are being used for sequencing as well as for providing new locations for binding heavy atom clusters by genetic insertion of cysteines on the ribosomal surface, according to accessibility data, obtained either chemically or by limited proteolysis. A nucleoprotein complex of protein HmaL1 and a fragment of the 23S rRNA was isolated from ribosomes. Chimeric complexes were reconstituted with E. coli ribosomal components, indicating rather high homolgy, despite the evolution distance.

The interplay between X-ray crystallography, neutron diffraction, image reconstruction, organo-metallic chemistry and biochemistry in structural studies of ribosomes

Crystals of ribosomes, their complexes with components of protein biosynthesis, their natural, mutated and modified subunits, have been subjected to X-ray and neutron crystallographic analyses. Electron miscroscopy and 3-dimensional image reconstruction, supported by biochemistry, genetic, functional and organo-metallic studies were employed for facilitating phasing of the crystallographic data. For example, a monofunctional multi heavy-atom cluster (undecagold) was designed for covalent and quantitative binding to ribosomes. The modified particles were crystallized isomorphously with the native ones. Their difference-Patterson maps contain indications for the usefulness of these derivatives for subsequent phasing. Models of the ribosome and its large subunit were reconstructed from tilt series of 2-dimensional sheets. The comparison of the various reconstructed images enabled an initial assessment of the reliability of these models and led to tentative assignments of several functional features. These include the presumed sites for binding mRNA and for codon-anticodon interactions, the path taken by the nascent protein chain and the mode for tRNA binding to ribosomes. These assignments assisted in the design of biologically meaningful crystal systems. The reconstructed models are being used to identify structural features in initial density maps derived from X-ray and neutron diffraction data.

Thermus thermophilus ribosomes for crystallographic studies

Three-dimensional crystal of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and thronyl- and seryl-tRNA synthesis from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 Å and 60 Å, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies.

Crystals of 70S ribosomes from thermophilic bacteria are suitable for X-ray amalysis at low resolution

Single crystals of 70S ribosomes from Thermus thermophilus and from the wild-type and a mutant of Bacillus stearothermophilus have been obtained. Using synchroton X-ray beam, these crystals diffract to 19 Å and 35 Å, respectively, and can be irradiated at temperatures of 110–120 K for days without noticeable decay. Diffraction data sets to 22 Å and to 33 Å with reasonable R scale were collected from the crystals of 70S from T. thermophilus. The cell dimensions ( a = b = 524 Å, c = 315 Å), the symmetry (P4 12 12 or P4 32 12) and the density (1.063 g/cm 3) of these crystals indicate that there is one 70S ribosome per asymmetric unit.

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 °C and examined in an X-ray synchrotron beam. The space group is P4 12 12 or P4 32 12 with unit cell dimensions of a = 510 A ̊ and c = 378 A ̊ . The diffraction patterns extend to better than 20 Å.

リボゾームの結晶

リボゾームの結晶

1] Studying ribosome structure by electron microscopy and computer-image processing

This chapter focuses on the study of ribosome structure by electron microscopy and computer-image processing. In the method of single-particle 3-D reconstruction the macromolecule is reconstructed in three dimensions from a large number of projections. In the variant of this method most viable for structure research, these projections come from different particles lying in, or brought into, different orientations providing a wide range of viewing directions. In principle, electron crystallographic techniques are capable of providing structural data on ribosomes. However, a resolution comparable to that obtained by single-particle reconstruction has not been achieved, mainly because large, well-ordered crystals of ribosomes or ribosomal subunits have been difficult to grow. The statistical significance of structural features in an averaged image can be assessed by applying standard statistical tests to the individual image elements. The theoretical resolution of a 3-D reconstruction, which depends on the number of projections, may be calculated for equal angular increments in both the single-axis and the conical-tilting schemes. Both formulas are derived by using Shannon's sampling theorem, which states in essence that two measurements must be available for each resolution elements.

Molecular structure determination of crystalline specimens in frozen aqueous solutions

Images were obtained of eukaryotic ribosome crystals, bladder membranes and gap junctions preserved in frozen aqueous solutions under conditions where either amorphous or crystalline ice is formed. Evaluation of these images by optical diffraction showed that specimens containing the largest open spaces were sensitive to ice crystal damage during freezing, whereas those containing the smallest open spaces were not. Projection maps were calculated from the images and compared to maps obtained from the same specimens at the same resolution in negative stain. Significant differences were apparent between each pair of maps. These were attributed to details being revealed of the RNA and protein (ribosomes) or the complete protein (membranes) when using frozen solutions, compared to just the hydrophilic surfaces when using stain. Thus the freezing method appears to provide the most complete and accurate descriptions of these structures.

Crystalline arrays of ribosomes in isolated pea chloroplasts

Abstract Transmission electron microscopy of chloroplasts isolated by osmotic lysis of pea leaf protoplasts has revealed crystalline arrays of ribosomal particles associated with the thylakoid membranes. Optical diffraction techniques have established the crystallinity of the arrays and an image‐enhancement technique has given an indication of ribosomal macrostructure. A model of crystal‐packing is presented. This apparently artefactual induction of ribosome crystals should provide a valuable approach towards the elucidation of the details of the structure of chloroplast ribosomes.

In vitro crystallization of ribosomes from chick embryos.

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.

Structure and evolution of ribosomes.

Ribosomes are the only cell organelles occurring in all organisms. E. coli ribosomes, which are the best characterized particles, consist of three RNAs and 53 proteins. All components have been isolated and characterized by chemical, physical and immunological methods. The primary structures of the RNAs and of all the proteins are known. Information about the secondary structure of the proteins derives from circular dichroism measurements and from secondary structure prediction methods. The tertiary structure is being studied by limited proteolysis, proton magnetic resonance and crystallization followed by X-ray analysis. Various methods are being used to elucidate the architecture of the ribosomal particle: three-dimensional image reconstruction of crystals of bacterial ribosomes and/or their subunits; immune electron microscopy; neutron scattering; protein-protein, protein-RNA and RNA-RNA crosslinking; total reconstitution of ribosomal subunits. The results from these studies yield valuable information on the architecture of the ribosomal particle. Many mutants have been isolated in which one or a few ribosomal proteins are altered or even deleted. The genetic and biochemical characterization of these mutants allows conclusions about the importance of these proteins for the function of the ribosome. Ribosomal proteins from various prokaryotic and eukaryotic species have been compared by two-dimensional gel electrophoresis, immunological methods, reconstitution and amino acid sequence analysis. These studies show a strong homology among prokaryotic ribosomal proteins but only a weak homology between proteins from prokaryotic and eukaryotic ribosomes. Comparison of the primary and secondary structures of the ribosomal RNAs from various organisms shows that the secondary structure of the RNA molecules has been strongly conserved throughout evolution.

Ribosome Structure Investigated by Digital Accumulation and Processing of Ribosome Tetramer Images

Electron microscopic observation of ribosomes, especially combined with 3-D reconstruction methods, may reveal structural relationships among components of the ribosome. In such studies it is necessary to average over a large number of specimen particles, to enhance the predominant structure while suppressing local irregularities of specimen preservation and staining, and to reduce the statistical insufficiencies of individual low-dose electron micrographs. One very successful method makes use of an average over the particles in a two-dimensional crystalline layer, but if the particles are not well organized, the resolution is reduced. Organization of crystalline arrays can be enhanced by computer methods, but if computational alignment is used it seems most profitable to avoid the restriction to crystalline spec- iment completely and work with isolated particles. A fixed orientation is still helpful; so in studying ribosomes, which lack their own symmetry, we have chosen to observe ribosome tetramers. The tetramers, units of the chick embryo ribosome crystal, present a fixed view in the electron micrograph.

X-ray study of the paracrystalline nature of crystallized 70Sribosome lamellae

If a mixed organic solvent is added to a ribosome solution and stored at 277 K in a crystallization chamber, thin lamellae of a few μm diameter can be obtained. Under the electron microscope, tetragonal face-centred macrocells of b = 40 and c = 55 nm are visible. These findings are substantiated by X-ray small- and wide-angle diffraction which leads to macrocells of b = 42 and c = 52 nm and establishes the third dimension of the macrocell with a = 42 nm. Mainly 0kl reflections appear. From their line widths, it follows that each lamella consists of at least 15 × 15 × 15 macrocells with small paracrystalline distortions gs - 1.5%, whilst their 6 × 8 × 12 subcells with a0 = 7.1, b0 = 5.3 and c0 = 4.3 nm have large fibrillary distortions in the bc plane, similar to the aggregation of microparacrystals in stretched and annealed polymers. With a new method, the folding root of the Q function projected on the b axis can be analysed. Two compounds are placed in each macrocell, each consisting of about 136 cylinders of about 2.0 nm diameter and 7 nm length. They are aligned to fibrils of different lengths orthogonal to the bc plane and occupy about 47% of the macrocell. The residual volume is filled up with an 'amorphous phase' as in semicrystalline polymers. A comprehensive study of the existing literature leads to the conclusion that the 'crystalline phase' consists of two 70S ribosome tetramers and that the cylinders are obviously parts of two-stranded helices, whilst most of the RNA proteins are in the 'amorphous phase'. The biological significance of the ribosome crystals and the paracrystalline sublattice is described in detail in the Discussion.

Cristallisation ribosomique et hibernation

SummaryIn the ovarian follicles of european lizards part of the ribosomes is constituted in crystalline sheets associated with membranes of smooth reticulum during the hibernation. These crystals belong to the plane P4 group. The process of formation is described and the ribosome crystals of the lizards are compared both with ribosome crystals of the chick embryo and with the ribosome arrangement observed in plants during the winter rest.The hypothesis about the physiological meaning of the ribosome crystallization are discussed.

Attachment of ribosome crystals to intracellular membranes

The attachment to membranes of ribosome crystals formed by cooling lizard oocytes and chick embryos has been investigated by electron microscopy of whole cells and by biochemical and structural experiments, using the cross-linking reagent glutaraldehyde. It was found that the crystalline ribosomes in both animals form only on the rough endoplasmic reticulum and nuclear envelope, that they bind to these membranes through one unique site on the large ribosomal subunit, that the bond between the large subunit and the site on the membrane is sensitive to the concentration of K +, but not of Mg 2+, and that this bond is selectively stabilized by mild treatment with glutaraldehyde. These results closely match those obtained from ribosomes in secretory cells, suggesting that there may be no difference between the two sets of ribosomes in their direct interaction with membranes. The glutaraldehyde reaction was used to obtain crystals and components from which the small subunits had been preferentially released. A comparison between small subunit depleted and normal crystals led to an estimate for the positions of the subunits over the membrane surface. The side-by-side subunit assignments, “S” and “L”, suggested previously (Unwin & Taddei, 1977; Unwin, 1977), were confirmed. It was deduced further that the crystalline ribosomes have the long axis of their small subunit approximately parallel to the membrane surface, and appear raised up from this surface because of interaction between their large subunits.

Ribosome crystallization in nuclei of chick embryos.

Ribosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.