Reticulocyte Ribosomes Research Articles

Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.

Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro.

Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.

Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems

Ribosome display is a technology for library selection and simultaneous molecular evolution in vitro. We present here a comparison between an optimized Escherichia coli system and different rabbit reticulocyte ribosome display systems, optimized in a number of parameters, as a coupled eukaryotic system had been suggested to result in high enrichment factors [He and Taussig (1997) Nucleic Acids Res. 25, 5132–5134]. With all systems, antibody scFv fragments, complexed to the ribosomes and the corresponding mRNA, were enriched by binding to their cognate antigen and enrichment was always dependent on the absence of a stop codon and the presence of cognate antigen. However, the efficiency of the E. coli ribosome display system was 100-fold higher than an optimized uncoupled rabbit reticulocyte ribosome display system, with separate in vitro transcription and translation, which was in turn several-fold more efficient than the reported coupled system. Neither the E. coli nor the rabbit reticulocyte ribosome display system was dependent on the orientation of the domains of an antibody scFv fragment or on the spacer sequence. In summary, we could not detect any intrinsic advantage of using a eukaryotic translation system for ribosome display.

Involvement of the amino acids outside the active-site cleft in the catalysis of ricin A chain.

The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869-4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 s ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 å resolution

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 Å resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Role of ribosomes in reinitiation of membrane insertion of internal transmembrane segments in a polytopic membrane protein.

The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.

Mapping of nuclease-sensitive sites in native reticulocyte ribosomes--an analysis of the accessibility of ribosomal RNA to enzymatic cleavage.

Treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium-dependent nuclease from Staphylococcus aureus resulted in cleavage of rRNA. The positions of the cleaved phosphodiester bonds were localised by primer extension and polyacrylamide gel electrophoresis. S. aureus nuclease-induced strand scissions were found in the 5'-domain of 18S rRNA and in domains II, IV and VI of 28S rRNA. The majority of the cleavage sites were located in eukaryote-specific expansion segments and only one cleavage site was found in a region suggested to be directly involved in ribosomal function. Treatment of the reticulocyte lysate with increasing amounts of S. aureus nuclease resulted in the introduction of new cleavage sites. However, even at the highest nuclease concentration used, large parts of the rRNAs were protected from nuclease digestion. Removal of translational components, by salt wash of isolated reticulocyte polysomes, exposed additional rRNA sequences to S. aureus nuclease cleavage. These sequences were found in the 3'-major domain of 18S rRNA and in domains II, IV, and V of 28S rRNA. These sites are located at the putative translational surface of the ribosome. The translational activity of the S. aureus nuclease-treated ribosomes, determined after addition of exogenous mRNA, was directly correlated to the extent of nuclease digestion of the ribosomes. However, the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease was not due to a preferential exclusion of damaged ribosomes from polysome formation. This suggests that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the mRNA.

Determination of Eukaryotic Peptidyltransferase Activity by Pseudo-First-Order Kinetic Analysis

We have developed anin vitrosystem for the determination of peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA–poly(U)–80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate (k3) constant is identical to the catalytic rate constant (kcat) of peptidyltransferase. Thisk3of peptidyltransferase is equal to 2.9 min−1at 37°C. Moreover, the ratiok3/Ks, which is an accurate measure of peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic peptidyltransferase.

Induction of a ribosome-inactivating protein upon environmental stress.

Transcripts of altered abundance in RNA from unstressed and 500 mm salt-shocked Mesembryanthemum crystallinum (common ice plant) were detected by reverse-transcription differential display (RT-DD). One transcript, Rip1, was of very low abundance in unstressed plants and was strongly induced by stress. RNA blot hybridizations showed strong induction and a diurnal rhythm of transcript abundance with a maximum each day around the middle of the light phase. Rip1 encodes a reading frame of 289 amino acids (molecular mass 32,652), RIP1, with homology to single-chain ribosome inactivating proteins (rRNA N-glycosidases). The deduced amino acid sequence is 31.7% identical to pokeweed antiviral protein RIP-C (overall similarity 66.5%) with highest identity in domains of documented functional importance. RT-DD also detected mRNA for pyruvate, orthophosphate dikinase (PPDK) which has already been shown to be stress-induced in the ice plant [16]. RIP1, expressed in Escherichia coli, showed rRNA N-glycosidase activity against ice plant and rabbit reticulocyte ribosomes. The induction of Rip1 coincides with the transition period during which global changes in translation lead to adaptation of the ice plant to salt stress.

A Reevaluation of the Cap-binding Protein, eIF4E, as a Rate-limiting Factor for Initiation of Translation in Reticulocyte Lysate

The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (approximately 75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported. The association of a small proportion of eIF4E with the ribosome fraction appears to be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate.

Point mutations in the Hydrophobic C-Terminal Region of Ricin A Chain Indicate that Pro250 Plays A Key Role in Membrane Translocation

A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps.

Point mutations in the Hydrophobic C-Terminal Region of Ricin A Chain Indicate that Pro250 Plays A Key Role in Membrane Translocation

A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps.

Mechanism of protein synthesis inhibition by didemnin B in vitro.

The cytotoxic and immunosuppressive marine depsipeptide didemnin B is a potent inhibitor of protein biosynthesis in intact cells. Here, didemnin B is shown to inhibit protein synthesis in vitro during the elongation cycle, by preventing eukaryotic elongation factor 2-(eEF-2-) dependent translocation. No inhibition of aminoacyl-tRNA delivery or of peptidyltransferase activity is observed. Didemnin B stimulates eEF-1 alpha-dependent aminoacyl-tRNA binding to rabbit reticulocyte ribosomes, and eEF-1 alpha is required for inhibition of the subsequent translocation of phenylalanyl-tRNA(Phe) from the A- to the P-site. These observations suggest that didemnin B prevents translocation by stabilizing aminoacyl-tRNA bound to the ribosomal A-site, similar to the antibiotic kirromycin, and consistent with the known affinity of didemnins for elongation factor eEF-1 alpha [Crews et al. (1994) J. Biol. Chem. 269, 15411]. Unlike kirromycin, didemnin B does not prevent peptide bond formation, so inhibition is observed only at the translocation step. Inhibition of translocation by didemnin B is attenuated by increasing concentrations of eEF-2.

The barley 60 kDa jasmonate‐induced protein (JIP60) is a novel ribosome‐inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.

Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis.

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[Ser]Sec did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA[Ser]Sec.

Ebulin 1, a nontoxic novel type 2 ribosome-inactivating protein from Sambucus ebulus L. leaves.

A novel type 2 ribosome-inactivating protein (RIP) that we named ebulin 1 has been isolated from leaves of Sambucus ebulus L. (Caprifoliaceae). In vitro ebulin 1 strongly inhibited protein synthesis by rabbit reticulocyte lysates, rat brain, and rat liver cell-free systems but did not affect in vitro plant nor bacterial protein synthesis. Ebulin 1 is composed of two subunits, a catalytic A subunit (M(r) 26,000) and a D-galactose-binding lectin B subunit (M(r) 30,000). Amino-terminal amino acid sequence homology revealed the novelty that the ebulin 1 A-chain shares a high degree of homology not with the A-chain of other type 2 RIPs but rather with the Cucurbitaceae type 1 RIP briodin S and the anti-human immunodeficiency virus type I proteins trichosanthin and TAP 29. Upon treatment with acid aniline the rRNA from ebulin 1-treated rabbit reticulocyte ribosomes released the RNA fragment which is diagnostic of RIP catalytic action. Ebulin 1 was nontoxic to mice up to 2 mg/kg of body weight and did not inhibit protein synthesis in cultured NHC human epithelial cells which are highly sensitive to ricin.

Role of cytoplasmic mRNP proteins in translation

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per mol of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA ≈ 2:1, mol/mol), this protein promotes the translation process. In free mRNPs (p50:mRNA ≈ 4:1, mol/mol) this protein is a translational repressor, a component of the above mentioned mRNA repressor/activator regulatory system.

Structural Studies on the Eukaryotic Ribosome

We have studied the eukaryotic ribosome from two vertebrate species (rabbit reticulocyte and chick embryo ribosomes) in several different electron microscopic preparations (Fig. 1a-d), and we have applied image processing methods to two of the types of images. Reticulocyte ribosomes were examined in both negative stain (0.5% uranyl acetate, in a double-carbon preparation) and frozen hydrated preparation as single-particle specimens. In addition, chick embryo ribosomes in tetrameric and crystalline assemblies in frozen hydrated preparation have been examined. 2D averaging, multivariate statistical analysis, and classification methods have been applied to the negatively stained single-particle micrographs and the frozen hydrated tetramer micrographs to obtain statistically well defined projection images of the ribosome (Fig. 2a,c). 3D reconstruction methods, the random conical reconstruction scheme and weighted back projection, were applied to the negative-stain data, and several closely related reconstructions were obtained. The principal 3D reconstruction (Fig. 2b), which has a resolution of 3.7 nm according to the differential phase residual criterion, can be compared to the images of individual ribosomes in a 2D tetramer average (Fig. 2c) at a similar resolution, and a good agreement of the general morphology and of many of the characteristic features is seen.Both data sets show the ribosome in roughly the same ’view’ or orientation, with respect to the adsorptive surface in the electron microscopic preparation, as judged by the agreement in both the projected form and the distribution of characteristic density features. The negative-stain reconstruction reveals details of the ribosome morphology; the 2D frozen-hydrated average provides projection information on the native mass-density distribution within the structure. The 40S subunit appears to have an elongate core of higher density, while the 60S subunit shows a more complex pattern of dense features, comprising a rather globular core, locally extending close to the particle surface.

A special repressor/activator system controls distribution of mRNA between translationally active and inactive mRNPs in rabbit reticulocytes

Translation of free mRNPs and polyribosomal mRNPs from rabbit reticulocytes was studied in a rabbit reticulocyte and wheat germ cell-free systems. It has been shown that the translation efficiency of polyribosomal mRNPs and the mRNA isolated from the particles is nearly the same in both systems. At the same time, free mRNPs' translatability, which is high in the homologous cell-free system, is very low in the system from wheat germs. Translation efficiency of free mRNPs in the wheat germ system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results testify to the existence of some special repressor/activator system which controls the distribution of mRNA between free mRNPs and polyribosomes in rabbit reticulocytes.

Three-dimensional reconstruction of mammalian 40 S ribosomal subunit

The small (40 S) subunit from rabbit reticulocyte ribosomes has been reconstructed from electron micrographs of a negatively stained single-particle specimen to a resolution of 3.85 nm. The reconstruction reveals a morphology consisting of a broad wedge-shaped head structure set atop a quasi-cylindrical body. Distinctive features recognized in two-dimensional projections, such as the beak, back lobes, and feet, can now be localized in three dimensions. By reference to a recent reconstruction of the monomeric 80 S ribosome we can identify the interface and exterior surfaces of the subunit, thus enabling more detailed functional interpretations.