Biomedical subjects

M Radermacher

Publications and source records attributed to M Radermacher.

At least 19 recordsLinked to original sources

PubMed1998

Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.

BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity. RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein. CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

Bacterial Proteins↗

PubMed1998

Structural characterization of a dynein motor domain.

Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule's minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of seven to eight globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin.

Adenosine Triphosphate↗

PubMed1998

[Autofluorescence characteristics of lipofuscin components in different forms of late senile macular degeneration].

BACKGROUND: Lipofuscin is the main fluorophore of the human fundus. Because lipofuscin is the result of the accumulation of metabolic debris in pigmentepithelial cells (RPE), the autofluorescence can be interpreted as a clinical sign for the metabolic activity of the RPE. In order to get informations of RPE-function in different types of late AMD, the autofluorescence patterns in patients with late AMD were analyzed. MATERIAL AND METHOD: A prospective examination of the fundus-autofluorescence of 64 eyes of 52 patients with different types of late AMD was performed using a confocal scanning-laser-opthalmoscope. The autofluorescence images were categorized in respect to the type of late AMD according to the opthalmoscopic and fluoresceine-angiographic findings. RESULTS: Reduced autofluorescence was found in the centre of occult (78.6%) and classic (100%) choroidal neovascularisations (NV) as well as in the occult NV of RPE detachments. A loss of autofluorescence was related to the RPE free area of RPE-tears (100%) and to RPE-atrophy (88.9%) with sometimes increased autofluorescence at the rim. Increased autofluorescence could be seen at the surface of RPE-detachments (71.4%), in the area of the shrink age of RPE in RPE-tears (100%) as well as at RPE-proliferations in small occult NV (100%). Disciforme scars showed variable patterns of autofluorescence. CONCLUSION: The autofluorescence of the RPE can be analyzed clinically with the described method. Different patterns of autofluorescence could be revealed in different types of late AMD. Increased autofluorescence was found in lesions with proliferative or phagocytotic metabolic activity of the RPE like RPE-detachments, shrinked RPE in RPE-tears or occult NV with RPE-proliferations. The reduced autofluorescence in occult or classical choroidal NV can be interpreted as a sign of decompensation of the RPE and was also seen in areas with RPE-loss.

Aged↗

PubMed1997

Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor.

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large ( approximately 80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.

Animals↗

PubMed1997

Low-dose automated electron tomography: a recent implementation.

Low-dose automated tomography has been implemented on a 400-kV JEOL intermediate voltage electron microscope. Instrumentation and procedures for automatic tomographic series data collection are described. Difficulties encountered and ways to overcome them are discussed. A low-dose tomographic projection series of a triad junction of frog sartorius muscle was semiautomatically collected and a 3-D reconstruction of this organelle was made.

Animals↗

PubMed1997

Clinical and genetic evidence for autosomal dominant North Carolina macular dystrophy in a German family.

PURPOSE: To describe a German family with clinical and genetic evidence of autosomal dominant North Carolina macular dystrophy. METHODS: Twenty-six individuals from a five-generation family from northern Germany were investigated clinically. In addition, we performed genetic linkage analyses using polymorphic markers from proximal 6q. RESULTS: The affected family members showed clinical abnormalities consistent with North Carolina macular dystrophy including multiple drusen, choroidal neovascularization in one patient, and geographic atrophy in elderly patients. The DNA analyses demonstrated significant linkage to the North Carolina macular dystrophy locus on chromosome 6q14-q16.2. CONCLUSION: Our findings provide strong evidence of a German pedigree with an autosomal dominant macular dystrophy manifesting with clinical abnormalities consistent with North Carolina macular dystrophy.

Adolescent↗

PubMed1997

Ryanodine receptors: structure and macromolecular interactions.

Ryanodine receptors (RyRs), a class of intracellular calcium release channels, are the largest ion channels known. Recently, cryoelectron microscopy and image reconstructions of isolated receptors have shown that most of the protein mass forms a porous, multidomain cytoplasmic assembly. Evidence is mounting that suggests that the cytoplasmic assembly communicates with the transmembrane regions over distances of 100 or greater. RyRs are centrally important in excitation-contraction coupling, which occurs at specialized regions where the sarcoplasmic reticulum, containing the RyRs, and the plasma membrane/transverse-tubule system form junctions. Numerous proteins are present at these junctions, some of which interact directly with the RyR.

Animals↗

PubMed1995

A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome.

The ribosome is formed by assembly of proteins and nucleic acids, and synthesizes proteins according to genetic instructions in all organisms. Many of the biochemical steps of this fundamental process are known, but a detailed understanding requires a well-defined structural model of the ribosome. Electron microscopy combined with image reconstruction of two-dimensional crystals or single ribosomes has been the most promising technique, but the resolution of the resulting models has been insufficient. Here we report a 25-A reconstruction of the ribosome from Escherichia coli, obtained by combining 4,300 projections of ice-embedded single particles. Our new reconstruction reveals a channel in the small ribosomal subunit and a bifurcating tunnel in the large subunit which may constitute pathways for the incoming message and the nascent polypeptide chain, respectively. Based on these new findings, a three-dimensional model of the basic framework of protein synthesis is presented.

Bacterial Proteins↗

PubMed1995

Three-dimensional architecture of the skeletal muscle ryanodine receptor.

Recent advances in determining the three-dimensional architecture of the skeletal muscle ryanodine receptor/calcium release channel (RyR) by cryo-electron microscopy and three-dimensional reconstruction are discussed. The tetrameric receptor is characterized by a large 4-fold symmetric cytoplasmic assembly that consists of many domains separated by solvent-containing crevices and holes. Experimental evidence suggests that at least one regulatory ligand, calmodulin, binds to sites on the cytoplasmic assembly that are at least 10 nanometers from the transmembrane channel.

Calcium Channels↗

PubMed1995

Three-dimensional reconstruction of mammalian 40 S ribosomal subunit embedded in ice.

A platform-like structure, which appears equivalent to the platform or lobe structure of the 30 S subunit of the eubacterial ribosome, is observed in the reconstruction of the small 40 S ribosomal subunit from images of ice-embedded particles. This cup-shaped structure, 15.0 nm in side length and 13.5 nm wide at its rim, extends obliquely upward on the back of the subunit. Other previously characterized features of the 40 S subunit can readily be identified: the head with its prominent beak structure, the body with its two back lobes expressed as relatively small-scale features, and the two widely separated feet that comprise the base of the subunit.

Animals↗

PubMed1994

Three-dimensional reconstruction from random projections: orientational alignment via Radon transforms.

A method for alignment of projections with unknown projecting directions towards a three-dimensional reference has been developed. The technique has been applied to the three-dimensional reconstruction from images of a frozen-hydrated preparation of 50S ribosomal subunits from Escherichia coli recorded in the electron microscope. The algorithm as used here combines the Single Exposure Conical Reconstruction Technique (SECReT) with a three-dimensional orientation search. The algorithm allows for the refinement of a reconstruction obtained with SECReT by refinement of the true projection angles, and by the inclusion of projections with a priori unknown random orientation. With model data it is demonstrated that the algorithm works reliably even for signal-to-noise ratios lower than 1.

Algorithms↗

PubMed1994

Cryo-electron microscopy and three-dimensional reconstruction of the calcium release channel/ryanodine receptor from skeletal muscle.

The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 degrees rotation about an axis in the plane of the specimen grid. The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca(2+)-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.

Animals↗

PubMed1993

Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent-extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

Animals↗

PubMed1992

Three-dimensional reconstruction of single particles negatively stained or in vitreous ice.

The random-conical reconstruction method has been highly successful in three-dimensional imaging of macromolecules under low-dose conditions. This article summarizes the different steps of this technique as applied to molecules prepared with negative staining or vitreous ice, and sketches out the current directions of development. We anticipate that by using new instrumental developments, transfer function correction and computational refinement techniques, a resolution in the range of 7-10 A could ultimately be achieved.

Calcium Channels↗

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↗

PubMed1992

Three-dimensional reconstruction of single particles embedded in ice.

Single particles embedded in ice pose new challenges for image processing because of the intrinsically low signal-to-noise ratio of such particles in electron micrographs. We have developed new techniques that address some of these problems and have applied these techniques to electron micrographs of the Escherichia coli ribosome. Data collection and reconstruction follow the protocol of the random-conical technique of Radermacher et al. [J. Microscopy 146 (1987) 113]. A reference-free alignment algorithm has been developed to overcome the propensity of reference-based algorithms to reinforce the reference motif in very noisy situations. In addition, an iterative 3D reconstruction method based on a chi-square minimization constraint has been developed and tested. This algorithm tends to reduce the effects of the missing angular range on the reconstruction, thereby facilitating the merging of random-conical data sets obtained from differently oriented particles.

Algorithms↗

PubMed1991

Interfacial energies and surface-tension forces involved in the preparation of thin, flat crystals of biological macromolecules for high-resolution electron microscopy.

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1 degree, and perhaps less than 0.2 degrees, over areas as large as 1 micron2 or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.

Adsorption↗

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↗