Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

Interaction of 16S rRNA With Protein S4 - a first step to visualization of the assembly of the Escherichia Coli 30S Ribosomal Subunit by Stem

1986 ◽  
Vol 44 ◽  
pp. 296-297
Author(s):  
V. Mandlyan ◽  
G. T. Oostergetel ◽  
J. S. Wall ◽  
J. F. Hainfeld ◽  
M. Boublik
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Large Angle ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Dark Field ◽  
Dimensional Structure ◽  
Radius Of Gyration ◽  
Field Mode ◽  
Transmission Electron

Understanding the mechanism of ribosome assembly and involvement in protein synthesis can be greatly facilitated by elucidation of its three-dimensional structure. The conformation, topography, and Interactions of ribosomal constituent proteins and RNAs can be directly studied by dedicated high resolution scanning transmission electron microscopy (STEM). The high (80%) efficiency in collection of scattered electrons in the dark-field mode makes 1t possible to visualize freezedried unstained specimens at low radiation dose (le/Å2); this minimizes many artifacts inherent in conventional transmission electron microscopy (staining, air-dry1ng, and radiation damage). In addition, the linear proportionality of the large-angle elastically scattered electrons to specimen mass thickness can be used for quantitative determination of molecular weight, mass distribution, and calculation of the apparent radius of gyration (RG), a parameter closely related to the threedimensional structure of the macromolecule.

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.

Imaging of platinum-shadowed macromolecular complexes in dark field

1984 ◽  
Vol 42 ◽  
pp. 146-147
Author(s):  
D.W. Andrews ◽  
F.P. Ottensmeyer
Keyword(s):  
Thin Film ◽  
Three Dimensional ◽  
Average Diameter ◽  
Dark Field ◽  
Bright Field ◽  
Field Mode ◽  
Macromolecular Complexes ◽  
Average Mass ◽  
Topological Features ◽  
Projection Images

Shadowing with heavy metals has been used for many years to enhance the topological features of biological macromolecular complexes. The three dimensional features present in directionaly shadowed specimens often simplifies interpretation of projection images provided by other techniques. One difficulty with the method is the relatively large amount of metal used to achieve sufficient contrast in bright field images. Thick shadow films are undesirable because they decrease resolution due to an increased tendency for microcrystalline aggregates to form, because decoration artefacts become more severe and increased cap thickness makes estimation of dimensions more uncertain.The large increase in contrast provided by the dark field mode of imaging allows the use of shadow replicas with a much lower average mass thickness. To form the images in Fig. 1, latex spheres of 0.087 μ average diameter were unidirectionally shadowed with platinum carbon (Pt-C) and a thin film of carbon was indirectly evaporated on the specimen as a support.

Electron crystallographic determination of the 3-D structure of staurolite at atomic resolution

1991 ◽  
Vol 49 ◽  
pp. 540-541
Author(s):  
Kenneth H. Downing ◽  
Hu Meisheng ◽  
Hans-Rudolf Went ◽  
Michael A. O'Keefe
Keyword(s):  
High Resolution ◽  
Three Dimensional ◽  
High Resolution Electron Microscopy ◽  
Atomic Resolution ◽  
Dimensional Structure ◽  
Structure Information ◽  
Resolution Electron ◽  
Scattering Effects ◽  
High Resolution Images ◽  
Effects Of Radiation

With current advances in electron microscope design, high resolution electron microscopy has become routine, and point resolutions of better than 2Å have been obtained in images of many inorganic crystals. Although this resolution is sufficient to resolve interatomic spacings, interpretation generally requires comparison of experimental images with calculations. Since the images are two-dimensional representations of projections of the full three-dimensional structure, information is invariably lost in the overlapping images of atoms at various heights. The technique of electron crystallography, in which information from several views of a crystal is combined, has been developed to obtain three-dimensional information on proteins. The resolution in images of proteins is severely limited by effects of radiation damage. In principle, atomic-resolution, 3D reconstructions should be obtainable from specimens that are resistant to damage. The most serious problem would appear to be in obtaining high-resolution images from areas that are thin enough that dynamical scattering effects can be ignored.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

The three-dimensional structure of frozen-hydrated left- and right-handed forms of bacterial flagellar filaments from an identical serotype

1986 ◽  
Vol 44 ◽  
pp. 298-299
Author(s):  
S. Trachtenberg ◽  
D. J. DeRosier
Keyword(s):  
Ionic Strength ◽  
Basal Body ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Protein Species ◽  
Liquid Environment ◽  
Bacterial Flagellum ◽  
Left And Right ◽  
Rotary Motor ◽  
Helical Parameters

The bacterial cell is propelled through the liquid environment by means of one or more rotating flagella. The bacterial flagellum is composed of a basal body (rotary motor), hook (universal coupler), and filament (propellor). The filament is a rigid helical assembly of only one protein species — flagellin. The filament can adopt different morphologies and change, reversibly, its helical parameters (pitch and hand) as a function of mechanical stress and chemical changes (pH, ionic strength) in the environment.

Revealing gross three-dimensional structure in the SEM

1987 ◽  
Vol 45 ◽  
pp. 656-657
Author(s):  
Sterling P. Newberry
Keyword(s):  
Freeze Drying ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Small Object ◽  
Final Solution ◽  
Dimensional Representation ◽  
X Ray ◽  
Three Dimensional Representation ◽  
Freeze Dried ◽  
Critical Point Drying

The beautiful three dimensional representation of small object surfaces by the SEM leads one to search for ways to open up the sample and look inside. Could this be the answer to a better microscopy for gross biological 3-D structure? We know from X-Ray microscope images that Freeze Drying and Critical Point Drying give promise of adequately preserving gross structure. Can we slice such preparations open for SEM inspection? In general these preparations crush more readily than they slice. Russell and Dagihlian got around the problem by “deembedding” a section before imaging. This some what defeats the advantages of direct dry preparation, thus we are reluctant to accept it as the final solution to our problem. Alternatively, consider fig 1 wherein a freeze dried onion root has a window cut in its surface by a micromanipulator during observation in the SEM.

Three-dimensional structure of a regular bacterial surface layer: The HPI-layer of Deinococcus radiodurans

1983 ◽  
Vol 41 ◽  
pp. 438-439
Author(s):  
W. Baumeister ◽  
M. Hahn ◽  
W.O. Saxton
Keyword(s):  
Outer Membrane ◽  
Protein Interactions ◽  
Deinococcus Radiodurans ◽  
Bacterial Species ◽  
Hexagonal Lattice ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Protein Protein Interactions ◽  
Bacterial Surface ◽  
Freeze Dried

Regularly organized surface (RS) layers are a feature common to many bacterial species; they are clearly more abundant than was anticipated even a few years ago. The RS-layers are believed to fulfil a variety of functions in the interaction between the cell and its environment (see e.g. [1]). The so-called HPI-layer of the radiotolerant bacterium Deinococcus radiodurans is a typical example of such a layer: It is composed of a single polypeptide species (Mr 105 kDa) arranged on a hexagonal lattice to form a network that covers the entire surface of the bacterium; it is associated with the outer membrane via hydrophobic protein-protein interactions.Isolated HPI-layer sheets, released from the outer membrane by detergent treatment, have been studied in the electron microscope making extensive use of the present arsenal of preparation techniques: negative staining, (auro- thio)glucose embedding, freeze-dried/unstained, freeze-dried/metal shadowed etc.Because of the notorious problem of lattice imperfections image processing usually followed the strategy of correlation averaging as outlined in some detail elsewhere.

Optimum Detector Arrangements in the Stem for Thick Objects

1976 ◽  
Vol 34 ◽  
pp. 548-549
Author(s):  
H. Rose ◽  
J. Fertig
Keyword(s):  
Signal To Noise Ratio ◽  
Dark Field ◽  
Imaging Method ◽  
Bright Field Image ◽  
Field Mode ◽  
Maximum Information ◽  
Subjective Impression ◽  
Specific Object ◽  
High Resolution Images ◽  
Pass Through

Radiation damage is the main obstacle in achieving high resolution images of biological objects. Therefore it is desirable to indicate that imaging method which yields maximum information about a specific object for a given number N0 of incident electrons. The amount of useful information is determined by the signal to noise ratio. Applying difference techniques the contrast in the STEM can be varied arbitrarily (l). Although the subjective impression of the image quality may be improved by such a procedure the amount of information available will not be changed. Most of the present day STEMs operate in the dark field mode using an annular detector which collects all electrons scattered out of the illumination cone. The corresponding incoherent bright field image is obtained from the electrons which pass through the hole of the dark field detector.

scholarly journals 3-D Imaging of Crystals at Atomic Resolution

1994 ◽  
Vol 332 ◽  
Author(s):  
M.A. O'keefe ◽  
K.H. Downing ◽  
H-R. Wenk ◽  
Hu Meisheng
Keyword(s):  
Mechanical Stability ◽  
Three Dimensional ◽  
Inorganic Materials ◽  
Three Dimensions ◽  
Dimensional Structure ◽  
Structure Factors ◽  
Potential Map ◽  
Structure Determinations ◽  
Unit Cells ◽  
High Resolution Images

ABSTRACTElectron crystallography has now been used to investigate the structures of inorganic materials in three dimensions. As a test of the method, amplitudes and phases of structure factors were obtained experimentally from high resolution images of staurolite taken in a number of different projections. From images in five orientations, a three-dimensional Coulomb potential map was constructed with a resolution of better than 1.4Å. The map clearly resolves all the cations (Al,Si,Fe) in the structure, and all of the oxygen atoms. This method promises great potential for structure determinations of small domains in heterogeneous crystals which are inaccessible to x-ray analysis. Three-dimensional structure determinations should be possible on small domains only approximately 10 unit cells wide, and may resolve site occupancies in addition to atom positions. Given a microscope stage with a suitable range of tilt and enough mechanical stability, the method could also be applied to small crystalline particles larger than about 50Å to 100Å. In addition, it may be possible to apply the method to derive the two-dimensional structure of periodic defects.

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