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.

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

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.

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.

Structural Analysis of Filaments Using the STEM

2000 ◽  
Vol 6 (S2) ◽  
pp. 862-863
Author(s):  
M. N. Simon ◽  
B. Y. Lin ◽  
J. S. Wall
Keyword(s):  
Scanning Transmission Electron Microscope ◽  
Digital Data ◽  
Mass Analysis ◽  
Biological Origin ◽  
Biotechnology Research ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Different Types ◽  
Scanning Transmission ◽  
History Of

The Scanning Transmission Electron Microscope (STEM) facility at BNL is an NIH Biotechnology Research Resource and as such is available to users with suitable projects, free of charge. Currently, many of our users' projects involve studying the structures of a variety of filaments. Most of these are of biological origin, although a couple involve conducting polymers. The STEM has a long history of being used to study different types of filaments and resolving controversies about their structure.The mainstay of the STEM is mass analysis on unstained, isolated, freeze-dried samples. On these, the STEM can collect in-focus digital data directly. In a scan (8 sec. in real time) of a sample, at each of 512x512 picture elements (pixels), the number of electrons scattered into two annular detectors is recorded. For each pixel, the number of scattered electrons is directly proportional to the mass thickness in that pixel.

Scanning Transmission EM (STEM) of dynein ATPases

1987 ◽  
Vol 45 ◽  
pp. 798-799
Author(s):  
Kenneth A. Johnson ◽  
Silvio P. Marchese-Ragona ◽  
Joseph S. Wall
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Transmission Electron Microscopy ◽  
Electron Scattering ◽  
Scanning Transmission Electron Microscopy ◽  
Mass Analysis ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Lines Of Evidence

The structure and molecular weight of the microtubule-dependent ATPase, dynein, was first established by scanning transmission electron microscopy (STEM) of dynein isolated from the cilia of Tetrahymena. It was shown that dynein consists of three globular heads joined to a common base by three slender, flexible strands. The globular heads have a diameter of 10-12 nm and the strands are 24 nm in length, such that the particles are 35 nm overall. Mass analysis by integration of electron scattering intensities in the STEM established a molecular weight of 1.9 million, with each head contributing 420,000. Several lines of evidence suggested that the base anchors the dynein to the A-tubule and the three independent, globular heads interact with the B-tubule of the adjacent outerdoublet in an ATP-dependent reaction to produce a force for sliding.

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.

Advances in Stem Instrumentation for Biology

1990 ◽  
Vol 48 (1) ◽  
pp. 362-363
Author(s):  
J. S. Wall
Keyword(s):  
Scanning Transmission Electron Microscope ◽  
Carbon Films ◽  
Specimen Preparation ◽  
Material Deposition ◽  
Active User ◽  
Percent Improvement ◽  
Scanning Transmission ◽  
The Moment ◽  
Film Substrate ◽  
High Level

The forte of the Scanning transmission Electron Microscope (STEM) is high resolution imaging with high contrast on thin specimens, as demonstrated by visualization of single heavy atoms. of equal importance for biology is the efficient utilization of all available signals, permitting low dose imaging of unstained single molecules such as DNA.Our work at Brookhaven has concentrated on: 1) design and construction of instruments optimized for a narrow range of biological applications and 2) use of such instruments in a very active user/collaborator program. Therefore our program is highly interactive with a strong emphasis on producing results which are interpretable with a high level of confidence.The major challenge we face at the moment is specimen preparation. The resolution of the STEM is better than 2.5 A, but measurements of resolution vs. dose level off at a resolution of 20 A at a dose of 10 el/A2 on a well-behaved biological specimen such as TMV (tobacco mosaic virus). To track down this problem we are examining all aspects of specimen preparation: purification of biological material, deposition on the thin film substrate, washing, fast freezing and freeze drying. As we attempt to improve our equipment/technique, we use image analysis of TMV internal controls included in all STEM samples as a monitor sensitive enough to detect even a few percent improvement. For delicate specimens, carbon films can be very harsh-leading to disruption of the sample. Therefore we are developing conducting polymer films as alternative substrates, as described elsewhere in these Proceedings. For specimen preparation studies, we have identified (from our user/collaborator program ) a variety of “canary” specimens, each uniquely sensitive to one particular aspect of sample preparation, so we can attempt to separate the variables involved.

High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes

1995 ◽  
Vol 108 (3) ◽  
pp. 1117-1125
Author(s):  
A. Moscatelli ◽  
C. Del Casino ◽  
L. Lozzi ◽  
G. Cai ◽  
M. Scali ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Nicotiana Tabacum ◽  
Pollen Tube ◽  
High Molecular Weight ◽  
Pollen Tubes ◽  
Biochemical Properties ◽  
Bovine Brain ◽  
Atp Binding ◽  
Heavy Chains ◽  
Dynein Heavy Chains

Nicotiana tabacum pollen tubes contain two high molecular weight polypeptides (about 400 kDa), which are specifically expressed during pollen germination and pollen tube growth in BK medium. The high molecular weight doublet resembles the dynein heavy chains in some biochemical properties. Sedimentation profiles of pollen tube extracts show that the high molecular weight bands have sedimentation coefficients of 22 S and 12 S, respectively. ATPase assay of sedimentation fractions shows an activity ten times higher when stimulated by the presence of bovine brain microtubules in fractions containing the 22 S high molecular weight polypeptide. Both these high molecular weight polypeptides can bind microtubules in an ATP-dependent fashion. A mouse antiserum to a synthetic peptide reproducing the sequence of the most conserved ATP-binding site among dynein heavy chains recognized the two high molecular weight polypeptides. Therefore these polypeptides have sequences immunologically related to the ATP binding sites of dynein heavy chains.

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