Computer averaging of electron micrographs of 40S ribosomal subunits.

An enhanced lateral view of the 40S ribosomal subunit of HeLa cells has been obtained by computer averaging of single particles visualized in the electron microscope. Application of crystallographic criteria to independent averages shows that the reproducibility of the result is comparable to that obtained for thin, stained protein crystals by conventional Fourier filtration methods.

Computer-averaged views of the 70 S monosome from Escherichia coli.

The prokaryotic (70 S) monosome, composed of a roughly hemispherical 50 S large subunit and an elongate 30 S small subunit, appears in the electron micrograph in only a few common views representing the small number of preferred orientations assumed by the particle. Two of these, termed O and L views, have previously been characterized as the overlap and non-overlap projections; a third view, which we term the R view, represents the other endpoint of a rotational continuum with the overlap or O view. Tilt studies enabled us to calibrate this range as spanning approximately 50 degrees. The disjunct set of L views was averaged, and the reproducible resolution was determined to be 1/3.5 nm-1. The combined sets of O and R views were analyzed by correspondence analysis, and a continuous "rotation series" of subaverages was obtained. Interpretation of the views in the light of what is known about the morphologies of the individual subunits allows a general picture of the mutual fit of the subunits in the monosome to be conceived.

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.

Visualization of protein-nucleic acid interactions involved in the in vitro assembly of the Escherichia coli 50 S ribosomal subunit.

Protein-nucleic acid interactions which occur during Escherichia coli 50 S ribosomal subunit assembly between 23 S rRNA, 5 S rRNA and a complete set of 34 L-proteins were monitored by high resolution scanning transmission electron microscopy (STEM). This approach made it possible to visualize and quantitatively analyze conformational changes induced in the rRNAs during E. coli 50 S ribosomal subunit assembly. The reconstituted RNA-protein complexes, the control 23 S rRNA and native 50 S subunits were characterized by their mass and morphology. Association of 23 S rRNA with the first assembly protein, L24, led to the formation of a distinct nucleus of mass ("cluster") on the filamentous and loosely coiled molecule of the 23 S rRNA. This structural feature was preserved and enhanced in 23 S rRNA after its association with the rest of the early assembly proteins, namely L3, L20, L13, L4 and L22. Since the above proteins, with the exception of L3, bind to the 5' end of the 23 S rRNA, the cluster seems to be formed predominantly by interactions of L24, L13, L20, L22 and L4 with this segment of the 23 S rRNA molecule. Association with the rest of the primary binding proteins (L2, L23, L9, L1), which interact with the 3' end of the 23 S rRNA, appears to result in the formation of a second mass center. Binding of additional proteins led to the formation of compact particles with an apparent similarity to the 50 S subunit. However, particles with defined structural features characteristic of the native 50 S subunit requires the interaction of both 23 S rRNA and 5 S rRNA with all of the L-proteins. STEM image analysis demonstrated that 50 S subunit reconstitution proceeds by the immediate folding of the 23 S rRNA into a single mass center followed by the formation of a second mass center. These mass centers merge into one central body, which is gradually enhanced and decorated with structural elements characteristic of the 50 S subunit in the latter stages of assembly.

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging.

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

Three-dimensional reconstruction of the 30 S ribosomal subunit from randomly oriented particles.

Electron micrographs show the small (30 S) subunit of Escherichia coli ribosomes lying in a wide range of positions on the specimen support, related by rotation principally around the long axis of the particle. Through correspondence analysis, a multivariate statistical method that distinguishes the major factors accounting for interimage variance, the (aligned) views of the randomly oriented particles were ordered and grouped according to tilt angle. Views so grouped were then averaged and used as input to a three-dimensional reconstruction program. The particle reconstructed from nine averaged projections spanning a 160 degrees rotational range has a resolution of 5 nm in planes perpendicular to the long axis of the particle and approximately 3 nm in the direction of the long axis. It is somewhat asymmetrical and quite compact; its most conspicuous feature is the "platform" that wraps partially around the middle of the subunit.

Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12.

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit.

The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy. This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps. 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure. The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A. Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend. While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17. With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A. Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa. The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance. Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others. The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20. The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA. However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis.

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.

Electron microscopic study of free and ribosome bound rRNAs from E. coli.

The morphology of ribosomal subunits, nucleoprotein cores, and free rRNAs from Escherichia coli has been studied by two high resolution electron microscopic techniques. Conventional transmission electron micrographs showed unfolding of 30S and 50S ribosomal subunits with increasing depletion of proteins. With dedicated (0.3 nm resolution) scanning transmission electron microscopy we were able to visualize unstained freeze dried ribosomal subunits and free rRNAs without artifacts of staining and structural distortion by air drying. By this technique, we have also determined the mass of individual ribosomal particles and rRNA molecules. While the ribosomal subunits displayed their characteristic structural features and mass, free rRNAs appeared unfolded and polydisperse. These results provide direct evidence for distinct conformational differences between free rRNAs and native ribosomal subunits of E. coli.

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy.

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.

Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy.

Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles. Variations in composition, relative molecular mass (Mr) and shape were observed both in the monosomes and in the ribosomal subunits. None of these changes can be resolved visually; they can be evaluated only by computer processing. The variations in relative mass and shape monitored by values of radius of gyration (RG) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity. The highest activity was found in monosomes prepared from the standard 0.5 M NH4Cl wash. With increasing concentrations (up to 1.5 M) of NH4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH4Cl. The most striking effects were observed in ribosomal particles washed with 0.1 M NH4Cl. The 70S monosomes and the 30S subunits attained maximum Mr and RG values (2660 kDa and 76 A, and 990 kDa and 75 A, respectively), which were greater than the theoretical values, while the activity was minimal (approximately 12%). The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (approximately 1600 kDa and 68 A).

Role of MR imaging in the management of injuries in professional football players.

This article discusses the effectiveness of MR imaging in evaluating the injuries of both upper and lower extremities in the professional football player. Topics include bone, joint and soft tissue disorders, and injuries resulting from overuse or trauma of the shoulder, elbow, forearm, and wrist; the pelvis and hip, lower extremity muscle and tendon, knee, and ankle.

Clinical examination of the shoulder complex.

A comprehensive and systematic approach to physical examination of the shoulder is described, with particular emphasis on findings pertinent to an athletic population. The format consists of initial impression, inspection, palpation, range of motion and strength testing, stability assessment, special tests, vascular examination, and general physical examination.