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T Wagenknecht

Publications and source records attributed to T Wagenknecht.

At least 19 recordsLinked to original sources
PubMed1992

Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum.

The native structure of the calcium release channel (ryanodine receptor) from rabbit skeletal muscle has been analyzed in two dimensions from electron micrographs of frozen hydrated specimens. Within a resolution of 3.0 nm there is excellent agreement between the structure as seen in vitreous water and in negative stained specimens. Features seen in the three-dimensional reconstruction of the negatively stained channel can be identified in the projection of the unstained receptor.

Animals↗
PubMed1991

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed.

Acetyltransferases↗
PubMed1990

Cryoelectron microscopy of frozen-hydrated alpha-ketoacid dehydrogenase complexes from Escherichia coli.

The native architectures of the pyruvate and 2-oxoglutarate dehydrogenase complexes have been investigated by cryoelectron microscopy of unstained, frozen-hydrated specimens. In pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex the transacylase (E2) components exist as 24-subunit, cube-shaped assemblies that form the structural cores of the complexes. Multiple copies (12-24) of the alpha-ketoacid dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components bind to the surface of the cores. Images of the frozen-hydrated enzyme complexes do not appear consistent with a symmetric arrangement of the E1 and E3 subunits about the octahedrally symmetric E2 core. Often the E1 or E3 subunits appear separated from the surface of the E2 core by 3-5 nm, and sometimes thin bridges of density appear in the gap between the E2 core and the bound subunits; studies of subcomplexes consisting of the E2 core from 2-oxoglutarate dehydrogenase complex and E1 or E3 show that both E1 and E3 are bound in this manner. Images of the E2 cores isolated from pyruvate dehydrogenase complex appear surrounded by a faint fuzz that extends approximately 10 nm from the surface of the core and likely corresponds to the lipoyl domains of the E2.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Three-dimensional reconstruction of native Androctonus australis hemocyanin.

A sample of native 4 x 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 degree and 0 degree tilt. The three-dimensional reconstruction method from "Single-exposure, random conical tilt series" was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 degree views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 x 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.

Animals↗
PubMed1989

Three-dimensional reconstruction of mammalian 40 S ribosomal subunit.

The small (40 S) subunit from rabbit reticulocyte ribosomes has been reconstructed from electron micrographs of a negatively stained single-particle specimen to a resolution of 3.85 nm. The reconstruction reveals a morphology consisting of a broad wedge-shaped head structure set atop a quasi-cylindrical body. Distinctive features recognized in two-dimensional projections, such as the beak, back lobes, and feet, can now be localized in three dimensions. By reference to a recent reconstruction of the monomeric 80 S ribosome we can identify the interface and exterior surfaces of the subunit, thus enabling more detailed functional interpretations.

Animals↗
PubMed1989

Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum.

The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.

Animals↗
PubMed1989

Three-dimensional reconstruction of the ribosome from Escherichia coli.

Three-dimensional image reconstruction has been applied to electron micrographs of noncrystalline, negatively stained ribosomes obtained from Escherichia coli. Several independent reconstructions all show an overall appearance resembling models that had been derived earlier by direct visual interpretation of electron micrographs. The reconstructed ribosomes show numerous structural details not recognized previously, some of which may be functionally significant. A large elongate cavity (approximately 8-nm long x 5-nm wide x 6-nm [maximal] deep) is present on the surface of the ribosome near the base of its stalk and is identifiable as a portion of a feature termed the interface canyon, which was detected in prior reconstructions of the large ribosomal subunit (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1107-1114). On the back of the ribosome, near the base of the central protuberance, is a hole leading to the interface canyon, which likely represents an exit site for the elongating polypeptide produced during protein biosynthesis. The exposed portion of the interface canyon appears well suited to bind two tRNA molecules in a configuration that is consistent with biochemical and structural data on the mechanism of peptide bond biosynthesis.

Escherichia coli↗
PubMed1989

Variations of the three-dimensional structure of the Escherichia coli ribosome in the range of overlap views. An application of the methods of multicone and local single-cone three-dimensional reconstruction.

Electron microscopic techniques are among the most important tools for obtaining structural information of biological specimens. However, the three-dimensional (3D) structural analysis of asymmetrical specimens that do not form crystalline sheets has traditionally presented serious methodological obstacles to its accomplishment. One of the fundamental questions to be addressed in this type of structural study is in what way, and to what degree, does the 3D structural conformation depend on the orientation of the specimen with respect to the electron microscopic support films. As a step in studying this problem, we have analyzed the variations of the 3D structure of the Escherichia coli 70S monosome by performing four different 3D reconstructions of the 70S monosome from subsets of images in the so-called overlap range of views. These subsets were selected according to a multivariate statistical analysis performed on the total population of overlap-range specimen images. A certain amount of structural variability exists among the 3D reconstructions, although many of the main morphological characteristics, as the relative orientation between the ribosomal subunits, remain unchanged. We have also generalized the random conical reconstruction technique (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. J. Microsc. 146: 113-136) to include those cases where the specimen exhibits a rocking behavior with respect to the support. The resulting Multicone Reconstruction Technique has been applied to computer-generated images as well as the E. coli 70S monosome images from part of the overlap range of views.

Analysis of Variance↗
PubMed1988

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging.

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.

Anticodon↗
PubMed1988

Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12.

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

Escherichia coli↗
PubMed1988

Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli.

Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.

Computers↗
PubMed1988

Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli.

Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC. The method is applied to two sets of 204 and 177 images that show the 70S ribosome of Escherichia coli, in the range of overlap views as defined by A. Verschoor and co-workers, and to two sets of 480 and 496 images of the 50S subunit of E. coli depleted of L7/L12 proteins in the well-defined crown view. Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates. These classes appear to be related to different orientations on the specimen grid (in the case of the 70S particle) and to different conformational states (50S subunit).

Escherichia coli↗
PubMed1987

Scanning transmission electron microscopic study of alpha-ketoglutarate dehydrogenase complex from Escherichia coli.

The alpha-ketoglutarate dehydrogenase complex was resolved into its three component enzymes: alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase. Subcomplexes were prepared in vitro by incubating the resolved E2, a 24-subunit cube-shaped molecule, with E1 (dimeric). The morphology and mass of the subcomplexes were determined by scanning transmission electron microscopy of negatively stained and of freeze-dried specimens. Images of both negative stained and freeze-dried subcomplexes were consistent with E1 binding at or near the midpoints of the edges of the E2 molecule. Mass analysis of the freeze-dried specimen showed that at least 95% of E1 remains in the dimeric state (or as two closely juxtaposed monomers) when it binds to E2.

Escherichia coli↗
PubMed1987

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.

Escherichia coli↗
PubMed1987

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

We present a new reconstruction method that takes advantage of the fact that many biological macromolecular assemblies show a preferred orientation with respect to the plane of the specimen grid in the electron microscopic preparation. From one micrograph taken of such a specimen tilted by a large angle, a conical tilt series with random azimuthal angles can be extracted and used for a three-dimensional reconstruction. Our technique allows the determination of the molecular structure under low-dose conditions, which are not achievable with reconstruction methods that use conventional tilt series. The reconstruction method combines a number of existing image processing techniques with a newly developed weighted back-projection algorithm designed for three-dimensional reconstruction from projections taken with arbitrary projecting directions. The method is described as it was applied to the three-dimensional reconstruction of the structure of the 50S ribosomal subunit of Escherichia coli (E. coli).

Escherichia coli↗
PubMed1986

Role of excess lipoyl dehydrogenase in reconstituted alpha-ketoglutarate dehydrogenase complex of Escherichia coli.

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli can bind up to 12 dimers of dihydrolipoyl dehydrogenase (E3) besides those already present. Maximal activity does not increase, however, when surplus E3 is present. This observation was previously interpreted to mean that the excess enzyme is inactive. We have now determined that if the reactions catalyzed by E3 are made rate-limiting, the excess E3 functions equivalently to that in the native complex.

Dihydrolipoamide Dehydrogenase↗