Ultrastructure of chromatin. II. Three-dimensional reconstruction of isolated fibers

1991 ◽  
Vol 99 (1) ◽  
pp. 107-114
Author(s):  
C.L. Woodcock ◽  
B.F. McEwen ◽  
J. Frank
Keyword(s):  
Three Dimensional ◽  
Carbon Support ◽  
Search Method ◽  
Necturus Maculosus ◽  
Peripheral Location ◽  
Solid Models ◽  
Dimensional Reconstruction ◽  
Peak Search ◽  
Tilt Series ◽  
Long Range Ordering

Electron-microscope tomography has been used to reconstruct isolated, negatively stained chromatin fibers from Necturus maculosus erythrocytes. Tilt series micrographs from +70 degrees to −70 degrees at 5 degrees intervals were obtained, allowing a reconstruction resolution of 3.3 nm for fibers lying parallel to the tilt axis. The fibers were found to be flattened in the plane of the carbon support, and also stained differentially according to the distance from the carbon. A number of methods of presenting the three-dimensional information were explored. Especially useful was an automatic peak search method for locating putative nucleosome positions coupled with the production of a computer-generated model. Other valuable techniques included the generation of projection stereograms and construction of solid models. A peripheral location of nucleosomes in the chromatin fiber was indicated, and helical arrangements of nucleosomes were observed over short regions. However, no long-range ordering of nucleosomes was apparent. The extent to which this lack of order may be the result of events occurring during the preparation of chromatin for electron microscopy is discussed.

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.

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 a mammalian Z-band from skeletal muscle

1992 ◽  
Vol 50 (1) ◽  
pp. 542-543
Author(s):  
J. P. Schroeter ◽  
M. A. Goldstein ◽  
J. P. Bretaudiere ◽  
R. L. Sass
Keyword(s):  
Skeletal Muscle ◽  
Cross Sections ◽  
Three Dimensional ◽  
Square Lattice ◽  
Back Projection ◽  
Axial Filament ◽  
Dimensional Reconstruction ◽  
Rat Soleus Muscle ◽  
Tilt Series ◽  
Three Dimensional Models

We have completed 3-d reconstructions of several regions of the Z-band in relaxed rat soleus muscle using the method of weighted back projection on a tilt series from two different longitudinal sections. Various displays of the reconstructions were interpreted after corrections for section shrinkage and comparisons to three dimensional models. Examination of cross-sections of the reconstructed Z-bands reveal that the lattice is in the small square form. We have previously shown that this form of the Z-band lattice is predominate in relaxed skeletal muscle. The reconstructions reveal that cross-connecting Z-filaments are arranged in opposing pairs along the axial filament. Successive pairs of filaments are rotated by ninety degrees about the axial filament, thus generating the four-fold appearance seen in the projected small square lattice.

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 reconstruction of A Z band from rigor skeletal muscle

1994 ◽  
Vol 52 ◽  
pp. 308-309
Author(s):  
J.P. Schroeter ◽  
R.J. Edwards ◽  
M. A. Goldstein
Keyword(s):  
Cross Sections ◽  
Lattice Spacing ◽  
Three Dimensional ◽  
Section Thickness ◽  
Back Projection ◽  
Dimensional Reconstruction ◽  
Rat Soleus Muscle ◽  
Projection Techniques ◽  
Tilt Series ◽  
Muscle Section

We have produced three dimensional reconstructions of several regions of the Z band and the nearby I band in rat soleus muscle in rigor. The reconstructions were calculated using weighted back-projection techniques on electron micrograph tilt series taken about two orthogonal axes. Examination of cross-sections of the reconstructed Z bands reveal that the lattice is in the basket-weave form. We have previously shown that this form of the Z band lattice predominates in the muscle in rigor. The reconstructions reveal a dense net of cross-connecting Z-filaments in the Z band, which interconnect the axial filaments in a projected four-fold array.The Z band lattice spacing was measured as 27 +− 4 nm, consistent with values seen in electron micrographs of cross sections of this muscle. The Z spacing and the form of the lattice show little distortion from that observed in cross sections, despite a beam induced change in section thickness from ∼80 to 45 nm. This suggests that, as in previous Z band reconstructions in unstimulated muscle, section shrinkage in the Z band does not occur by means of a uniform collapse throughout the section.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Sign in / Sign up

Export Citation Format

Share Document