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Biomedical subjects

M Radermacher

Publications and source records attributed to M Radermacher.

At least 55 records · Page 3Linked to original sources
PubMed1988

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.

Escherichia coli↗
PubMed1988

Three-dimensional reconstruction of single particles from random and nonrandom tilt series.

To overcome the radiation damage-induced limitations to the resolution of three-dimensional reconstructions from electron microscopic tilt series, novel reconstruction schemes have been developed that require only a single exposure of the specimen. The tilt series collected with these methods have random projection directions. First, three-dimensional reconstruction techniques are described that are applicable to data obtained from tilt series with regular tilt geometry, followed by the extensions of these techniques to permit analysis of projection series with randomly spaced tilts. The main emphasis is placed on the weighted back-projection methods, which have recently been extended so as to be applicable to random tilt series. Besides a description of the algorithms, the complete procedure for a three-dimensional reconstruction from a single-exposure, random conical tilt series is explained, including the determination of the azimuthal angles, the alignment scheme for conical tilt series, the dependence of the achievable resolution on the number of projections for regular conical and single-axis geometries, and the method to calculate the actual resolution of two-dimensional image averages and of three-dimensional reconstructions using the phase residual and Fourier ring correlation criteria. Examples are given of biological specimens to which these three-dimensional reconstruction methods have been applied.

Algorithms↗
PubMed1988

Ultrastructure of the calcium release channel of sarcoplasmic reticulum.

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.

Animals↗
PubMed1987

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.

Escherichia coli↗
PubMed1987

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).

Escherichia coli↗
PubMed1986

Tomographic three-dimensional reconstruction of cilia ultrastructure from thick sections.

We have applied a computer-based tomographic technique to reconstruct the three-dimensional ultrastructure of newt lung cilia. Epon-embedded samples were cut into 0.25-micron-thick sections that were imaged at 1 MV with a high-voltage electron microscope. For the reconstruction shown, a tilt series of 53 micrographs was taken at tilt angles between -54 degrees and +50 degrees. The reconstruction was accomplished from these projections using a weighted back-projection algorithm. The 12-nm resolution of the reconstruction was sufficient to resolve the outer doublet and central pair microtubules, dynein arms, radial spokes, and central sheath structures. The reconstruction can be viewed from various angles and with appropriate parts cut away to reveal structural features of interest. The sense of depth in these views can be enhanced by stereo viewing of shaded surface images. From this reconstruction, we determined that newt lung cilia contain the more common triplet grouping of radial spokes.

Animals↗
PubMed1985

The layered organization of nucleosomes in 30 nm chromatin fibers.

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10-11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5-6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.

Animals↗
PubMed1985

Shape changes and polarization of cells migrating through tissue. A high-voltage electron microscope and computer graphics study of serial thick sections.

Structural changes of carcinoma cells and fibroblasts migrating through small spaces in the elastic-collagen reticulum of mouse peritoneum have been studied by high-voltage electron microscopy of serial thick sections and by computer graphics reconstruction of cell profiles. The change of shape profile of an individual cell, between serial sections is large and the distribution of organelles is very non-uniform and changes markedly between sections. Conclusions about adhesion, intercell contact, cell shape and polarization of cytoplasmic organelles could only be reached by assessing a complete set of serial sections. Our preliminary results suggest that interesting structural changes occur in both carcinoma cells and fibroblasts when migrating through this tissue.

Adenocarcinoma↗
PubMed1985

Use of nonlinear mapping in multivariate image analysis of molecule projections.

A nonlinear mapping algorithm has been designed for the statistical analysis of sets of images in electron microscopy. The algorithm is appropriate in situations where it is not isolated features that determine the clustering or trends in the data set but a combination of several simultaneously changing features. The combination of the nonlinear mapping algorithm with correspondence analysis offers a way of ordering a set of images according to the variations of a single feature, as well as according to the combined variation of several independent features. The application of the combined algorithms ranges from image selection for averaging procedures to the sorting of images, that inherently form a tilt series. These sorted images then can be used for the three-dimensional reconstruction of macromolecules.

Animals↗
PubMed1985

The structure of the stalk surface layer of a brine pond microorganism: correlation averaging applied to a double layered lattice structure.

The surface layer of the stalk of a prosthecate halophilic microorganism is a periodic array (space group p3m1) comprised of electron dense trimers with a centre to centre spacing of 9.0 nm. The structure is reminiscent of E. coli porin. We have demonstrated that the method of correlation averaging can be effectively used to separate overlapping lattices, and that this results in a higher fidelity reconstruction, when compared to the established method of Fourier filtration. Two statistical methods are used to determine the resolution of the correlation average as a function of the number of 'windows' averaged. (i) The phase residual of spatial frequencies in Fourier space is computed between independently obtained subaverages, and (ii) a new method of Q factor analysis examines the cumulative vector sum in Fourier space as a function of the number of windows averaged. Both methods give a resolution of 1/2.1 nm-1.

Bacteria↗
PubMed1984

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.

Escherichia coli↗
PubMed1984

Representation of three-dimensionally reconstructed objects in electron microscopy by surfaces of equal density.

A surface representation algorithm has been developed to visualize the results of three-dimensional reconstructions from projections in electron microscopy. It produces an easily recognizable image of the reconstructed particle and facilitates comprehension of the three-dimensional structure. Thus building of physical models can be avoided in many cases. If such a model is needed the algorithm can be used to establish the correct particle boundary in advance. Some further effort has been made to represent long fibres and visualize inner structures of objects, which are obscured by outer high-density regions. Shading may be used to enhance the depth perception from single views.

Animals↗
PubMed1983

Three-dimensional reconstruction of chromatin fibers.

We have obtained tilt series of thin sections of chromatin fibers embedded in araldite-epon and stained with uranyl acetate. The tilt series contain between 12 and 18 micrographs, spanning tilt ranges between 84 and 117 degrees. Reconstructions in three dimensions have been obtained by filtered back-projection from each tilt series. The reconstructions have been low-pass filtered in order to reduce the amount of noise. In the reconstructions it is possible to approximately localize the nucleosomes. In several regions they show a clear zigzag arrangement, but in other regions it is difficult to determine the sequence of the nucleosomes. In any case there is no clear indication of a solenoidal arrangement. We discuss the rules which may give rise to a 3-D arrangement of the nucleosomal zigzag.

Chromatin↗