Three-Dimensional Reconstruction of a Mitochondrion Based on a Thick Section Tilt Series Recorded in the HVEM

1980 ◽  
Vol 38 ◽  
pp. 46-47
Author(s):  
J. Frank ◽  
J. N. Turner ◽  
M. Marko ◽  
K. Asmus ◽  
D. F. Parsons
Keyword(s):  
Room Temperature ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Angular Range ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction ◽  
Tilt Series ◽  
Adult Cat ◽  
The Brain ◽  
Aortic Perfusion

A three-dimensional reconstruction of a mitochondrion has been made based on a tilt series recorded in our HVEM. Fig. 1 is a low magnification view of an axon in cat motor cortex showing the general cellular ultrastructure and the mitochondrion used in the reconstruction. Fig. 2 shows five of the images of the mitochondrion recorded over the angular range +27° to −30°. A total of nine images were used in the reconstruction. The brain of a normal adult cat was fixed by aortic perfusion using 4% buffered paraformaldehyde with sucrose at 4°C for 15 min. The brain was then removed and fixed overnight in glut-eraldehyde. Postfixation was carried out in 1% oso4 for one hour, and the sample was then dehydrated and embedded in epon. Half micron sections were stained in aqueous uranyl acetate for two hours at 50°C followed by Reynolds lead citrate for one hour at room temperature. The tilt series was recorded in our AEI EM7 at 1.0MeV using the Swann (1) double tilt stage.

Three-Dimensional Reconstruction of Two Forms of the Chaperonin GRO EL

1990 ◽  
Vol 48 (1) ◽  
pp. 258-259
Author(s):  
J.L. Carrascosa ◽  
G. Abella ◽  
S. Marco ◽  
M. Muyal ◽  
J.M. Carazo
Keyword(s):  
Three Dimensional ◽  
The Other ◽  
Two Dimensional ◽  
Series Method ◽  
Three Dimensional Reconstruction ◽  
Structural Components ◽  
E Coli ◽  
Dimensional Reconstruction ◽  
Morphogenetic Factors ◽  
Tilt Series

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.

Three-dimensional reconstruction of neurons in the brain by using X-ray micro-CT technology

2007 ◽  
Vol 58 ◽  
pp. S184 ◽  
Author(s):  
Haruo Mizutani ◽  
Yoshihiro Takeda ◽  
Atsushi Momose ◽  
Toshihisa Takagi
Keyword(s):  
Three Dimensional ◽  
Micro Ct ◽  
Three Dimensional Reconstruction ◽  
X Ray ◽  
Dimensional Reconstruction ◽  
Ct Technology ◽  
The Brain

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

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.

Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

1987 ◽  
Vol 45 ◽  
pp. 936-937
Author(s):  
Neng-Yu Zhang ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
Tom Obrig ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Uranyl Acetate ◽  
Reconstruction Method ◽  
Single Exposure ◽  
Electron Dose ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

Angular Calibration of Ribosomal Subuntts in Factor Space

1990 ◽  
Vol 48 (1) ◽  
pp. 462-463
Author(s):  
Minakhi Pujari ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Carbon Layer ◽  
Uranyl Acetate ◽  
Particle Analysis ◽  
Factor Space ◽  
Multivariate Statistical ◽  
Ribosomal Subunits ◽  
Layer Method ◽  
Dimensional Reconstruction ◽  
Number Of Particles

In single-particle analysis of macromolecule images with the electron microscope, variations of projections are often observed that can be attributed to the changes of the particle’s orientation on the specimen grid (“rocking”). In the multivariate statistical analysis (MSA) of such projections, a single factor is often found that expresses a large portion of these variations. Successful angle calibration of this “rocking factor” would mean that correct angles can be assigned to a large number of particles, thus facilitating three-dimensional reconstruction.In a study to explore angle calibration in factor space, we used 40S ribosomal subunits, which are known to rock around an axis approximately coincident with their long axis. We analyzed micrographs of a field of these particles, taken with 20° tilt and without tilt, using the standard methods of alignment and MSA. The specimen was prepared with the double carbon-layer method, using uranyl acetate for negative staining. In the MSA analysis, the untilted-particle projections were used as active, the tilted-particle projections as inactive objects. Upon tilting, those particles whose rocking axes are parallel to the tilt axis will change their appearance in the same way as under the influence of rocking. Therefore, each vector, in factor space, joining a tilted and untilted projection of the same particle can be regarded as a local 20-degree calibration bar.

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