Conformation of eukaryotic ribosomal RNAs

1983 ◽  
Vol 41 ◽  
pp. 592-593
Author(s):  
M. Boublik ◽  
G. Thornton ◽  
G. Oostergetel ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Molecular Weight ◽  
Mass Measurement ◽  
Carbon Film ◽  
Structural Complexity ◽  
Scanning Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Pure Nitrogen ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Partially Unfolded

Understanding the structural complexity of ribosomes and their role in protein synthesis requires knowledge of the conformation of their components - rRNAs and proteins. Application of dedicated scanning transmission electron microscope (STEM), electrical discharge of the support carbon film in an atmosphere of pure nitrogen, and determination of the molecular weight of individual rRNAs enabled us to obtain high resolution electron microscopic images of unstained freeze-dried rRNA molecules from BHK cells in a form suitable for evaluation of their 3-D structure. Preliminary values for the molecular weight of 28S RNA from the large and 18S RNA from the small ribosomal subunits as obtained by mass measurement were 1.84 x 106 and 0.97 x 106, respectively. Conformation of rRNAs consists, in general, of alternating segments of intramolecular hairpin stems and single stranded loops in a proportion which depends on their ionic environment, the Mg++ concentration in particular. Molecules of 28S RNA (Fig. 1) and 18S RNA (not shown) obtained by freeze-drying from a solution of 60 mM NH+4 acetate and 2 mM Mg++ acetate, pH 7, appear as partially unfolded coils with compact cores suggesting a high degree of ordered secondary structure.

Improved preservation of freeze-dried biological specimens

1992 ◽  
Vol 50 (1) ◽  
pp. 736-737
Author(s):  
Martha N. Simon ◽  
Beth Y. Lin ◽  
Joseph S. Wall
Keyword(s):  
Freeze Drying ◽  
Carbon Film ◽  
Scanning Transmission Electron Microscope ◽  
Carbon Substrate ◽  
Final Thickness ◽  
Thin Carbon ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Thin Carbon Film ◽  
Strong Attachment

Specimens prepared by the wet-film technique (injecting unstained biological specimens into a drop of buffer on a thin carbon substrate which has never seen air, washing extensively, blotting to a thin layer of liquid, plunging the grid into nitrogen slush, and freeze-drying overnight) then visualized in the scanning transmission electron microscope (STEM) usually have reasonably wel1-preserved structures. However, there is a certain variability from day to day and sometimes even from one area to another on a given grid. This can occur for different reasons which may be inextricably related. The thin carbon film can be non-uniform at the molecular level with hot spots for strong attachment of some specimens, a part of a biological specimen may attach strongly while the rest of it thrashes about in Brownian motion ruining any perceivable structure, and the final thickness of liquid before freeze-drying may vary slightly which may affect the preservation of the structure.

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.

scholarly journals Structure and molecular weight of the dynein ATPase

1983 ◽  
Vol 96 (3) ◽  
pp. 669-678 ◽  
Author(s):  
KA Johnson ◽  
JS Wall
Keyword(s):  
Molecular Weight ◽  
Unit Length ◽  
Attachment Site ◽  
Carbon Films ◽  
Bovine Brain ◽  
Mass Analysis ◽  
Uranyl Sulfate ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Cilia And Flagella

Dynein has been examined by scanning transmission electron microscopy (STEM). Samples of 30S dynein from tetrahymena cilia were applied to carbon films and either were freeze- dried and examined as unstained, unfixed specimens, or were negatively stained with uranyl sulfate. A totally new image of the dynein molecule was revealed showing three globular heads connected by three separate strands to a common base. Two of the heads appeared to be identical and exhibited a diameter of 10 nm while the third head was somewhat larger (approximately 12 nm). The overall length of the particle was 35 nm. Mass analysis, based upon the integration of electron scattering intensities for unstained particles, gave a molecular weight of 1.95 (+/-)0.24) megadaltons. Mass per unit length analysis was performed using bovine brain microtubules decorated with dynein under conditions where the dynein shows a linear repeat of 24 nm with seven dynein molecules surrounding a microtubule made up of 14 protofilaments. Undecorated microtubules gave a molecular weight per unit length of 21,000+/-1,900 daltons/A, compared to a value of 84,400+/-2,200 daltons/A for the fully decorated microtubules. Taken together, these data give a molecular weight of 2.17 (+/- 0.14) megadaltons per dynein molecule, in agreement with measurements on the isolated particles. Mass analysis of individual globular heads observed in isolated particles gave a molecular weight distribution with a mean of 416+/- 76 kdaltons. These data could also be viewed as the sum of two populations of head with two-thirds of the heads at approximately 400 kdaltons and one-third at approximately 550 kdaltons, although more precise data will be required to distinguish two classes of heads with confidence. The mass of the dynein-microtubule complex as a function of distance from the midline of the particle was analysed to distinguish which end of the dynein molecule was bound to the microtubule. The projected mass distribution was consistent with a model where the three dynein heads were oriented toward the microtubule and clearly not consistent with the opposite orientation. These data indicate that the three globular heads form the ATP-sensitive site in this heterologous dynein-microtubule system and suggest that the rootlike base of the dynein molecule forms the structural attachment site to the A-subfiber of the outer doublet in cilia and flagella. The structure and function of the dynein are dicussed in terms of these new results.

X-Ray Microanalysis Combined with Monte Carlo Simulation for the Analysis of Layered Thin Films: The Case of Carbon Contamination

2009 ◽  
Vol 15 (2) ◽  
pp. 99-105 ◽  
Author(s):  
Aldo Armigliato ◽  
Rodolfo Rosa
Keyword(s):  
Monte Carlo ◽  
Carbon Film ◽  
Sample Surface ◽  
Scanning Transmission Electron Microscope ◽  
Carbon Layer ◽  
Monte Carlo Code ◽  
X Ray ◽  
Transmission Electron ◽  
The Monte Carlo Method ◽  
Scanning Transmission

AbstractA previously developed Monte Carlo code has been extended to the X-ray microanalysis in a (scanning) transmission electron microscope of plan sections, consisting of bilayers and triple layers. To test the validity of this method for quantification purposes, a commercially available NiOx (x ∼ 1) thin film, deposited on a carbon layer, has been chosen. The composition and thickness of the NiO film and the thickness of the C support layer are obtained by fitting to the three X-ray intensity ratios I(NiK)/I(OK), I(NiK)/I(CK), and I(OK)/I(CK). Moreover, it has been investigated to what extent the resulting film composition is affected by the presence of a contaminating carbon film at the sample surface. To this end, the sample has been analyzed both in the (recommended) “grid downward” geometry and in the upside/down (“grid upward”) situation. It is found that a carbon contaminating film of few tens of nanometers must be assumed in both cases, in addition to the C support film. Consequently, assuming the proper C/NiOx/C stack in the simulations, the Monte Carlo method yields the correct oxygen concentration and thickness of the NiOx film.

Radial Mass Density Analysis of Unstained Stem Images

1985 ◽  
Vol 43 ◽  
pp. 318-319
Author(s):  
P.S. Furcinitti ◽  
J.F. Hainfeld ◽  
J.J. Lipka ◽  
J.S. Wall
Keyword(s):  
Signal To Noise Ratio ◽  
Mass Density ◽  
Unit Length ◽  
Large Angle ◽  
Scanning Transmission Electron Microscope ◽  
Outer Diameter ◽  
Biological Macromolecules ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Spot 5

The high contrast and signal-to-noise ratio inherent in the Scanning Transmission Electron Microscope (STEM) makes it possible to examine unstained, freeze-dried biological macromolecules. Since the large-angle, elastically scattered STEM signal is directly proportional to the specimen mass, molecular weight or mass per unit length determinations are possible for individual macromolecules. For objects which have cylindrical or spherical symmetry the resolution lost by sparse sampling (2 Å spot, 5 or 10 Å between pixels) may be regained by employing the “Vernier Sampling” method developed by Steven et al. to rebin the data on a finer grid. A projected mass distribution is then obtained for the average values of the mass per unit area on an axis perpendicular to the symmetry axis. Assuming the particle to consist of a set of concentric cylinders of varying density, a set of simultaneous equations can be solved for the mass density at each annular ring. Thus the outer diameter and the internal radial structure of complex macromolecules Can be determined.

Observation of the evolution of 55-atom icosahedral Ag clusters by coherent electron nanodiffraction in a UHV stem

1994 ◽  
Vol 52 ◽  
pp. 788-789
Author(s):  
J. Liu ◽  
G. E. Spinnler
Keyword(s):  
Carbon Film ◽  
Beam Current ◽  
Scanning Transmission Electron Microscope ◽  
Nano Particles ◽  
Ex Situ ◽  
Beam Current Density ◽  
Unstable State ◽  
Specimen Preparation ◽  
Probe Size ◽  
Scanning Transmission

The morphology of metallic nano particles fluctuates and rapidly changes under the influence of electron beam irradiation. Structural transformations of small Au particles and disordering of small Pt clusters have been reported. Although the existence of a quasimolten state in nano particles has been experimentally observed there are still many unanswered questions concerning the onset of this unstable state. The previous experiments were carried out in non-UHV microscopes and the particles were prepared ex situ. We report preliminary observations of the evolution of clean Ag nano particles in a UHV scanning transmission electron microscope (STEM).The experiments were performed on a UHV STEM (VG HB-501S), known by the acronym MIDAS (Microscope for Imaging, Diffraction and Analysis of Surfaces). The probe size used for nanodiffraction was approximately 1 nm in diameter with a beam current density of 104 A/cm2. Silver nano particles were formed by evaporating Ag onto a pre-cleaned thin (< 5 nm in thickness) carbon film inside the UHV specimen preparation chamber attached to the MIDAS column.x

Mass Measurement with the Stem-Biological Applications

1980 ◽  
Vol 38 ◽  
pp. 674-675
Author(s):  
J. Hainfeld ◽  
J. Wall
Keyword(s):  
Cross Sections ◽  
Mass Measurement ◽  
Large Angle ◽  
Scanning Transmission Electron Microscope ◽  
Atomic Weight ◽  
Single Type ◽  
Mass Thickness ◽  
Charge Dependence ◽  
Scanning Transmission ◽  
Annular Detector

The scanning transmission electron microscope (STEM) is well equipped to perform quantitative measurements on biological specimens. Fertig and Rose have shown that signal from the large angle annular detector in the STEM is essentially an incoherent superposition of the signals from independent scatterers. This means that for a sample composed of only a single type of atom, the large angle annular detector signal is directly porportional to mass thickness. For atoms close together in the periodic table, the Z3/2 (Z=nuclear charge) dependence of elastic scattering can usually be approximated as the atomic weight and again the signal will be nearly proportional to mass thickness. For atoms of widely differing Z, it will usually be necessary to rely on knowledge of specimen composition and experimentally determined scattering cross sections.

Contamination at Low Temperature

1977 ◽  
Vol 35 ◽  
pp. 558-559 ◽  
Author(s):  
J. Wall ◽  
J. Bittner ◽  
J. Hainfeld
Keyword(s):  
Low Temperature ◽  
Heavy Atom ◽  
Carbon Film ◽  
Scanning Transmission Electron Microscope ◽  
Dark Field ◽  
Transmission Electron ◽  
Surface Migration ◽  
Thin Carbon ◽  
Scanning Transmission ◽  
Atom Migration

Contamination of biological specimens has been a practical limitation for many years. It is especially noticeable in the dark field STEM (Scanning Transmission Electron Microscope) since contamination only occurs near the area irradiated by the beam and since we are most often observing very thin specimens (20 - 40 Å). Contamination can be greatly reduced by baking the specimen to 100°C in vacuum prior to observation, (1), but such heating may alter biological structures and cause heavy atom migration away from heavy atom staining sites.Since contamination results from surface migration (2), it might be eliminated by cooling the specimen to a sufficiently low temperature. The Brookhaven STEM (3).is equipped with a cold stage capable of attaining -170°C with less than 5 Å vibration. Contamination was observed as a function of temperature for a clean carbon film and for two biological specimens. The specimens were prepared on slotted Titanium grids covered by a holey carbon film with thin carbon (˜ 20 A) stretched across the holes.

Mass Measurement of Macromolecular Complexes with the Stem

1982 ◽  
Vol 40 ◽  
pp. 362-363
Author(s):  
J. S. Wall ◽  
J. F. Hainfeld
Keyword(s):  
Mass Measurement ◽  
Unit Length ◽  
Scanning Transmission Electron Microscope ◽  
Particle Identification ◽  
Thickness Variation ◽  
Specimen Preparation ◽  
Complex Object ◽  
Substrate Thickness ◽  
Random Errors ◽  
Scanning Transmission

Mass measurement with the Scanning Transmission Electron Microscope (STEM) is a technique with wide application which has been developed in several laboratories. It is especially useful for particle identification in micrographs or in measurements of mass per unit length and mass distribution within a complex object.The accuracy of mass measurements is determined by random errors (Counting statistics, substrate thickness variation) and systematic errors (biochemical degradation, salt, specimen preparation artifacts, radiation damage). Random errors can be estimated from previous studies and range from 1-10%.

Progress in Single Atom Microscopy

1977 ◽  
Vol 35 ◽  
pp. 78-79 ◽  
Author(s):  
M. Isaacson ◽  
D. Kopf ◽  
M. Utlaut
Keyword(s):  
Surface Science ◽  
Science Studies ◽  
Carbon Film ◽  
Scanning Transmission Electron Microscope ◽  
Single Atom ◽  
Spacing Distribution ◽  
Heavy Atoms ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
And Diffusion

We have indicated in previous publications that a scanning transmission electron microscope (STEM) capable of atomic resolution may be a useful instrument for surface science studies and, in particular, capable of reliably observing the distribution and diffusion of individual atoms on thin film substrates (1-3). In this paper we will present further observations relating to the characteristics of individual heavy atoms on low atomic number substrates.In figure 1, we show pair spacing distributions of individual uranium (1A) and silver (IB) atoms adsorbed to thin (˜20 Å thick) carbon film substrates. These distributions are obtained by measuring spacings between all pairs of atoms on many different fields of view (2). The two distributions for each atom type are for different specimens.(The spacing distribution is defined as the number of atoms/Å2 in an annulus of 1 Å width at a given distance from any atom.) For both the U and Ag distributions, there is a pronounced peak near 4-5 Å.

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