Three-Dimensional reconstruction of E. coli SecA at low resolution

Chaperonins are a class of proteins characterized by their role as morphogenetic factors. They trantsiently interact with the structural components of certain biological aggregates (viruses, enzymes etc), promoting their correct folding, assembly and, eventually transport. The groEL factor from E. coli is a conspicuous member of the chaperonins, as it promotes the assembly and morphogenesis of bacterial oligomers and/viral structures.We have studied groEL-like factors from two different bacteria:E. coli and B.subtilis. These factors share common morphological features , showing two different views: one is 6-fold, while the other shows 7 morphological units. There is also a correlation between the presence of a dominant 6-fold view and the fact of both bacteria been grown at low temperature (32°C), while the 7-fold is the main view at higher temperatures (42°C). As the two-dimensional projections of groEL were difficult to interprete, we studied their three-dimensional reconstruction by the random conical tilt series method from negatively stained particles.

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Challenges of three-dimensional reconstruction of ribonucleoprotein complexes from electron spectroscopic images - Reconstructing ribosomal RNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162727 ◽

1996 ◽

Vol 54 ◽

pp. 52-53

Author(s):

D.R. Beniac ◽

G.J. Czarnota ◽

T.A. Bartlett ◽

F.P. Ottensmeyer ◽

G. Harauz

Keyword(s):

Ribosomal Rna ◽

Correlation Functions ◽

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Mutual Correlation ◽

Ribosomal Subunits ◽

E Coli ◽

Ribonucleoprotein Complexes ◽

Dimensional Reconstruction

Transmission electron microscopy has been dominant in structural studies of the ribosome and its constituent ribonucleic acids and proteins. Ribosomal RNA (rRNA) has central importance in the architecture of this complex and in protein synthesis. Our work has entailed using electron spectroscopic imaging (ESI) to probe the tertiary structure of rRNA in situ in a prokaryote (Escherichia coli) and in a eukaryote (Thermomyces lanuginosus). ESI uses only electrons which have lost a specific amount of energy due to specific inner-shell ionisation interactions with the specimen to form an elemental map. In nucleoprotein complexes, a map of the phosphorus distribution represents primarily a projection of the phosphate backbone of the nucleic acid component. The visualisation of rRNA in situ in the intact ribosomal subunit by ESI was demonstrated almost a decade ago to be feasible. Our work on quantitative image analysis of ES images of E. coli and Th. lanuginosus ribosomal subunits has presented unique challenges and has resulted in new algorithmic developments generally applicable to such images. These innovations include a singular pretreatment procedure, the use of mutual correlation functions rather than cross correlation functions to reduce the effect of low spatial frequency components, and angular determination using iterative quaternion-assisted angular reconstitution to compute a three-dimensional reconstruction. These investigations have produced direct information regarding ribosomal rRNA localisation in the ribosomal subunits of E. coli and Th. lanuginosus, and the position of non-conserved sequences.

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Low resolution three dimensional reconstruction of CMEs using solar mass ejection imager (SMEI) data

10.1117/12.616329 ◽

2005 ◽

Cited By ~ 4

Author(s):

Bernard V. Jackson ◽

Andrew Buffington ◽

P. P. Hick ◽

Cindy X. Wang

Keyword(s):

Three Dimensional ◽

Three Dimensional Reconstruction ◽

Low Resolution ◽

Solar Mass ◽

Solar Mass Ejection Imager ◽

Dimensional Reconstruction ◽

Mass Ejection

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Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122733 ◽

1992 ◽

Vol 50 (1) ◽

pp. 466-467

Author(s):

Daniel Beniac ◽

George Harauz

Keyword(s):

Ribosomal Rna ◽

Three Dimensional ◽

Spectroscopic Imaging ◽

Electron Spectroscopic Imaging ◽

E Coli ◽

Microscopical Technique ◽

Quantitative Image ◽

Dimensional Reconstruction ◽

Image Averaging ◽

Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

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Methods for three-dimensional reconstruction of cellular components within a thick section

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141871 ◽

1986 ◽

Vol 44 ◽

pp. 18-21

Author(s):

J. Frank ◽

B. F. McEwen ◽

M. Radermacher ◽

C. L. Rieder

Keyword(s):

Tilt Angle ◽

Tomographic Reconstruction ◽

3D Structure ◽

Three Dimensional ◽

Thick Section ◽

Three Dimensional Reconstruction ◽

Axis Ratio ◽

Dimensional Reconstruction ◽

Cellular Components

The tomographic reconstruction from multiple projections of cellular components, within a thick section, offers a way of visualizing and quantifying their three-dimensional (3D) structure. However, asymmetric objects require as many views from the widest tilt range as possible; otherwise the reconstruction may be uninterpretable. Even if not for geometric obstructions, the increasing pathway of electrons, as the tilt angle is increased, poses the ultimate upper limitation to the projection range. With the maximum tilt angle being fixed, the only way to improve the faithfulness of the reconstruction is by changing the mode of the tilting from single-axis to conical; a point within the object projected with a tilt angle of 60° and a full 360° azimuthal range is then reconstructed as a slightly elliptic (axis ratio 1.2 : 1) sphere.

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Three-Dimensional Reconstruction of Native Androctonus Australis Hemocyanin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181014 ◽

1990 ◽

Vol 48 (1) ◽

pp. 452-453

Author(s):

Nicolas Boisset ◽

Jean-Christophe Taveau ◽

Jean Lamy ◽

Terence Wagenknecht ◽

Michael Radermacher ◽

...

Keyword(s):

Electron Microscopy ◽

Solid Body ◽

Body Surface ◽

Three Dimensional ◽

Staining Technique ◽

Three Dimensional Reconstruction ◽

Dimensional Reconstruction ◽

View Reconstruction ◽

Surface Representations ◽

Top View

Hemocyanin, the respiratory pigment of the scorpion Androctonus australis is composed of 24 kidney shaped subunits. A model of architecture supported by many indirect arguments has been deduced from electron microscopy (EM) and immuno-EM. To ascertain, the disposition of the subunits within the oligomer, the 24mer was submitted to three-dimensional reconstruction by the method of single-exposure random-conical tilt series.A sample of native hemocyanin, prepared with the double layer negative staining technique, was observed by transmisson electron microscopy under low-dose conditions. Six 3D-reconstructions were carried out indenpendently from top, side and 45°views. The results are composed of solid-body surface representations, and slices extracted from the reconstruction volume.The main two characters of the molecule previously reported by Van Heel and Frank, were constantly found in the solid-body surface representations. These features are the presence of two different faces called flip and flop and a rocking of the molecule around an axis passing through diagonnally opposed hexamers. Furthermore, in the solid-body surface of the top view reconstruction, the positions and orientations of the bridges connecting the half molecules were found in excellent agreement with those predicted by the model.

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