Adriana Verschoor

Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli

We present a new reconstruction method that takes advantage of the fact that many biological macromolecular assemblies show a preferred orientation with respect to the plane of the specimen grid in the electron microscopic preparation. From one micrograph taken of such a specimen tilted by a large angle, a conical tilt series with random azimuthal angles can be extracted and used for a three‐dimensional reconstruction. Our technique allows the determination of the molecular structure under low‐dose conditions, which are not achievable with reconstruction methods that use conventional tilt series. The reconstruction method combines a number of existing image processing techniques with a newly developed weighted back‐projection algorithm designed for three‐dimensional reconstruction from projections taken with arbitrary projecting directions. The method is described as it was applied to the three‐dimensional reconstruction of the structure of the 50S ribosomal subunit of Escherichia coli (E. coli).

Multivariate statistical analysis of ribosome electron micrographs: L and R lateral views of the 40 S subunit from HeLa cells

Abstract The morphology of the small (40 S) subunit of the eukaryotic ribosome from HeLa cells has been examined by single-particle averaging and multivariate image analysis applied to electron micrographs of negatively stained specimens. The use of multivariate image analysis allows different, independent components of the structural variation within the particles to be identified and separately studied. The largest component of variance for both lateral views (termed L and R) was the variation in the peripheral stain intensity. The second largest component of interparticle variation is due to changes in the particle appearance most likely associated with a change of orientation on the specimen film. Averages formed from particles falling within a small range of peripheral stain intensity allowed the changes in the projected structure to be studied as a function of local stain level. Visual observations of stain variation could be confirmed quantitatively. Significant differences were found between averages of particles in the L view and those in the R view. Multivariate image analysis of a mixed population of L and mirrored R particles showed that the differences consistently affect all particles. However, the R view increasingly resembles the L view as the overall level of stain is increased, in agreement with a model of partial stain immersion.

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three‐dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three‐dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.

Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli.

Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low‐dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC.

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

Publisher Summary 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 Shannons sampling theorem, which states in essence that two measurements must be available for each resolution elements.

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.

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.

Structure of Ribosomes and Their Components by Advanced Techniques of Electron Microscopy and Computer Image Analysis

High-resolution electron microscopy plays a leading role in the structural analysis of biological macromolecules and is the most direct method for obtaining detailed information on the morphology, topography of the components, and functional sites of ribosomes. Electron microscopy (EM) has been important also for the interpretation of data obtained by a variety of physico-chemical techniques (for references see Chambliss et al., 1980; Liljas, 1982; Wittmann, 1983) on ribosomal protein locations, relative protein-protein distances, and protein-RNA binding sites, data which are more valuable when mapped within a well-defined structural framework provided, at present, by EM.

Functional Site Determinations in Three Dimensions on Eukaryotic and Eubacterial Ribosomes

Immuno electron microscopy has been a powerful tool in efforts to map exposures of individual r-proteins and functional sites on the ribosome, in that it has provided direct visual identification of the contact between an antibody and its epitope, as seen in an electron micrograph of a single-particle preparation of ribosomes. However, the information has been only two-dimensional: the site is seen on a projection image of the particle. If it can be identified on several different projections -- representing different orientations of the ribosome -- some three-dimensional (3D) information can be deduced, but the localization can be only approximate until 3D reconstruction techniques are applied to the problem.