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

2018 ◽  
pp. 138-161
Author(s):  
M. RADERMACHER ◽  
T. WAGENKNECHT ◽  
A. VERSCHOOR ◽  
J. FRANK
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Three Dimensional Reconstruction ◽  
Single Exposure ◽  
Dimensional Reconstruction ◽  
Tilt Series

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.

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.

scholarly journals Three-dimensional reconstruction of the ribosome from Escherichia coli

1989 ◽  
Vol 55 (3) ◽  
pp. 455-464 ◽  
Author(s):  
T. Wagenknecht ◽  
J.M. Carazo ◽  
M. Radermacher ◽  
J. Frank
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction

Negative staining versus rotational evaporation

1983 ◽  
Vol 41 ◽  
pp. 598-599
Author(s):  
H. Schmiady ◽  
C. Kreuzfeldt ◽  
E. Reuber ◽  
B. Tesche
Keyword(s):  
Test Specimen ◽  
Staining Method ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Negative Staining ◽  
Positive Staining ◽  
Negative Stain ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Tilt Series

This comparative study demonstrates the salient differences between the two most common methods of contrasting subcellular particles, using the 40S ribosomal subunit from saccharomyces cerevisiae as test specimen because its socalled beak-like L (left) and R (right) particle projections can be unequivocally distinguished.The negative staining method produces images which are not artifact-free because it is a combination of fixation and chemical staining and, therefore, not neutral towards the object. For three-dimensional reconstruction it is unsatisfactory because:(1) The affinity of the support film to the negative stain solution differs so greatly that sometimes undesired positive staining effects occur.(2) The rendition of the structure depends on the embedding--that is, on the level of the negative stain; fluctuations in the level lead to a loss and/or change of the structure, and thus to difficulties in interpreting the stain distribution, especially when reconstruction of the object by means of tilt series is planned.

Three-dimensional reconstruction of mammalian 40 S ribosomal subunit

1989 ◽  
Vol 209 (1) ◽  
pp. 115-126 ◽  
Author(s):  
A. Verschoor ◽  
N.-Y. Zhang ◽  
T. Wagenknecht ◽  
T. Obrig ◽  
M. Radermacher ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction

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.

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