Morphology of escherichia coli ribosomes as obtained by high resolution shadow cast

1978 ◽  
Vol 36 (2) ◽  
pp. 240-241 ◽  
Author(s):  
B. Tesche ◽  
G.W. Tischendorf ◽  
G. Stöffler
Keyword(s):  
Freeze Drying ◽  
Three Dimensional ◽  
Optical Resolution ◽  
Single Particles ◽  
Three Dimensional Model ◽  
Rna Molecules ◽  
E Coli ◽  
Fine Grained ◽  
Shadow Casting ◽  
Shadow Cast

All components (54 proteins and 3 RNA molecules) of the E. coli ribosome have been identified and characterized by biochemical and biophysical means. The morphology of the ribosome and the structural arrangement of its components, however, is not yet unequivocally known. The reasons for this are that crystal arrangements could not be obtained and that the different preparations of single particles remain invariant; and that holds for different laboratories as well.We applied a preparation which has been demonstrated to be most gentle and hence most structure-preserving; that is, freeze-drying with shadow casting at -150°C. Furthermore, the evaporator is of such design that it produces very little heat and renders a thin, fine-grained tungsten layer. An electron optical resolution of 6 Å can be achieved. Fig. 1 shows a micrograph of a typical shadowgraph obtained with 70S ribosomes from which we attempt to derive a three-dimensional model of the ribosome.

scholarly journals Mutagenesis in the α3α4 GyrA Helix and in the Toprim Domain of GyrB Refines the Contribution of Mycobacterium tuberculosis DNA Gyrase to Intrinsic Resistance to Quinolones

2008 ◽  
Vol 52 (8) ◽  
pp. 2909-2914 ◽  
Author(s):  
Stéphanie Matrat ◽  
Alexandra Aubry ◽  
Claudine Mayer ◽  
Vincent Jarlier ◽  
Emmanuelle Cambau
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Primary Structure ◽  
Three Dimensional ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Intrinsic Resistance ◽  
E Coli

ABSTRACT The replacement of M74 in GyrA, A83 in GyrA, and R447 in GyrB of Mycobacterium tuberculosis gyrase by their Escherichia coli homologs resulted in active enzymes as quinolone susceptible as the E. coli gyrase. This demonstrates that the primary structure of gyrase determines intrinsic quinolone resistance and was supported by a three-dimensional model of N-terminal GyrA.

scholarly journals Three-Dimensional Model for the Human Cl−/HCO3− Exchanger, AE1, by Homology to the E. coli ClC Protein

2013 ◽  
Vol 425 (14) ◽  
pp. 2591-2608 ◽  
Author(s):  
Pamela Bonar ◽  
Hans-Peter Schneider ◽  
Holger M. Becker ◽  
Joachim W. Deitmer ◽  
Joseph R. Casey
Keyword(s):  
Three Dimensional ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
E Coli

3D Reconstruction of Proteasomes Using Random Conical Tilting

1990 ◽  
Vol 48 (1) ◽  
pp. 272-273
Author(s):  
R. Hegerl ◽  
G. Pfeifer ◽  
B. Dahlmann ◽  
W. Baumeister
Keyword(s):  
3D Reconstruction ◽  
Tilt Angle ◽  
Three Dimensional ◽  
Ammonium Molybdate ◽  
Two Dimensional ◽  
Single Particles ◽  
Three Dimensional Model ◽  
Cylinder Axis ◽  
Electron Dose ◽  
Image Pairs

Proteasomes isolated from the archaebacterium Thermoplasma acidophilum and negatively stained with ammonium molybdate appear in two preferred orientations on electron micrographs. Describing the particle roughly as cylinder-shaped, the orientations "end-on" and "side-on" are defined by the cylinder axis being normal and parallel to the specimen support, respectively. Two-dimensional averages are available from both views. The complexity of the particle, however, prevents an intuitive deduction of a three-dimensional model from these two views. Due to the occurence of two preferred orientations, the object is, in principle, well suited for a 3D reconstruction based on random conical tilting. The side-on view was used for 3D reconstruction for two reasons: First, two-dimensional averages led us to suppose that the particles do not suffer from rotations around their cylinder axis, and second, parallelism of the long cylinder axis and the specimen support promises to give a stable object inclination.The proteasomes were isolated and prepared for electron microscopy as described previously. Many pairs of micrographs were taken in a Philips EM 420 at a magnification of 36000 and an electron dose of about 2000 e/nm2. The first exposure of each pair shows the specimen with a tilt angle of about 60 deg, the second one is untilted (Fig. 1). Several image pairs were selected for computer processing using a favourable distribution of particles and acceptable focus conditions as selection criteria. After digitizing corresponding areas (2048 by 2048 pixels, pixel size 15 μm = 0.42 nm at object level) in each pair using an Eikonix 1412 camera, small images of single particles were extracted from the untilted image and aligned with respect to translation and rotation. Knowing the tilt angle and tilt axis azimuth, position and rotation parameters of the particles could be transferred to the tilted image thus enabling the extraction of a set 227 of projections ready for a three-dimensional back projection (Fig. 2). All computation was done with the EM-system.

scholarly journals Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2)

2020 ◽  
pp. jbc.RA120.015189
Author(s):  
Isadora A. Oliveira ◽  
Diego Allonso ◽  
Tácio V. A. Fernandes ◽  
Daniela M.S. Lucena ◽  
Gustavo T. Ventura ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Expression And Purification ◽  
Three Dimensional Model ◽  
Hexosamine Biosynthetic Pathway ◽  
E Coli ◽  
Cellular Processes ◽  
Catalytically Active ◽  
Dynamics Simulations

Glycoconjugates play a central role in several cellular processes and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein, and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

scholarly journals Updates on enzymatic and structural properties of human glutamine: fructose-6-phosphate amidotransferase 2 (hGFAT2)

2020 ◽  
Author(s):  
Isadora A. Oliveira ◽  
Diego Allonso ◽  
Tácio V. A. Fernandes ◽  
Daniela M. S. Lucena ◽  
Gustavo T. Ventura ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Three Dimensional ◽  
Dimensional Model ◽  
Expression And Purification ◽  
Three Dimensional Model ◽  
Hexosamine Biosynthetic Pathway ◽  
E Coli ◽  
Cellular Processes ◽  
Catalytically Active ◽  
Dynamics Simulations

AbstractGlycoconjugates play a central role in several cellular processes and alteration in their composition is associated to human pathologies. The hexosamine biosynthetic pathway is a route through which cells obtain substrates for cellular glycosylation, and is controlled by the glutamine: fructose-6-phosphate amidotransferase (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer, however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in E. coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerase fructose-6-phosphate into glucose-6-phosphate even in presence of equimolar amounts of glutamine, in an unproductive glutamine hydrolysis. Structural analysis of the generated three-dimensional model rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations show such loop as the most flexible portion of the protein, which interacts with the protein mainly through the interdomain region, and plays a key role on conformational states of hGFAT2. Altogether, our study provides the first comprehensive set of data on the structure, kinetics and mechanics of hGFAT2, which will certainly contribute for further studies focusing on drug development targeting hGFAT2.

Structural Homology between Prokaryotic and Eukaryotic Ribosomes

1977 ◽  
Vol 35 ◽  
pp. 416-417
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Ribosomal Proteins ◽  
Critical Evaluation ◽  
Three Dimensional ◽  
Structural Complexity ◽  
Carbon Support ◽  
Uranyl Acetate ◽  
Three Dimensional Model ◽  
Ribonucleic Acids ◽  
E Coli ◽  
70S Ribosome

The structural complexity of ribosomes is evident from the large number of their constituents - ribosomal proteins and ribonucleic acids. The simplest known prokaryotic (70S) ribosome from E. coli is composed of 53 different proteins and three ribonucleic acid molecules (23S, 16S and 5S RNA). Eukaryotic (80S) ribosomes also consist of three RNAs (28S, 18S and 5S) but contain a higher number of proteins, approximately 70. The mutual interactions of ribosomal proteins and RNAs determine the final structure and thus the biological activity of the ribosome. As expected for particles with the above composition, ribosomes and both ribosomal subunits have no symmetry or repetition in structure.Electron micrographs of ribosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) show distinct contours and electron dense pattern. Critical evaluation of different views of the two-dimensional projections of the ribosomal contours can provide a reasonable basis for proposing a three-dimensional model of the ribosome.

scholarly journals Three Dimensional Analytical Separation of Grain Boundary and Surface Scatterings in Polycrystalline Metal Films in the Case of Non Cubic Grains

1981 ◽  
Vol 9 (2) ◽  
pp. 125-130 ◽  
Author(s):  
C. R. Tellier ◽  
C. R. Pichard ◽  
A. J. Tosser
Keyword(s):  
Grain Boundaries ◽  
Three Dimensional ◽  
Approximate Model ◽  
Metal Films ◽  
Three Dimensional Model ◽  
Polycrystalline Metal ◽  
Fine Grained ◽  
Thin Polycrystalline ◽  
Analytical Separation ◽  
Approximate Expressions

Analytical approximate expressions for the resistivity and its temperature coefficient of thin polycrystalline metal films have been derived by considering separately the contributions of the grain-boundaries perpendicular to thex-,y- andz-axes. Provided that the grain-boundaries act as moderately efficient scatterers reasonable deviations from the three-dimensional model are obtained; an approximate model then seems convenient with which to perform the calculations of the strain coefficients of such fine-grained films.

scholarly journals Modelling of E. coli distribution in coastal areas subjected to combined sewer overflows

2013 ◽  
Vol 68 (5) ◽  
pp. 1123-1136 ◽  
Author(s):  
Mauro De Marchis ◽  
Gabriele Freni ◽  
Enrico Napoli
Keyword(s):  
Three Dimensional ◽  
Drainage System ◽  
Integrated Approach ◽  
Propagation Model ◽  
Integrated Modelling ◽  
Combined Sewer Overflows ◽  
Quality Model ◽  
Three Dimensional Model ◽  
E Coli ◽  
The Impact

Rivers, lakes and the sea were the natural receivers of raw urban waste and storm waters for a long time but the low sustainability of such practice, the increase of population and a renewed environmental sensibility increased researcher interest in the analysis and mitigation of the impact of urban waters on receiving water bodies (RWB). In Europe, the integrated modelling of drainage systems and RWB has been promoted as a promising approach for implementing the Water Framework Directive. A particular interest is given to the fate of pathogens and especially of Escherichia coli, in all the cases in which an interaction between population and the RWB is foreseen. The present paper aims to propose an integrated water quality model involving the analysis of several sewer systems (SS) discharging their polluting overflows on the coast in a sensitive marine environment. From a modelling point of view, the proposed application integrated one-dimensional drainage system models with a complex three-dimensional model analysing the propagation in space and time of E. coli in the coastal marine area. The integrated approach was tested in a real case study (the Acicastello bay in Italy) where data were available both for SS model and for RWB propagation model calibration. The analysis shows a good agreement between the model and monitored data. The integrated model was demonstrated to be a valuable tool for investigating the pollutant propagation and to highlight the most impacted areas.

scholarly journals Simulated Diffusion of Phosphorylated CheY through the Cytoplasm of Escherichia coli

2005 ◽  
Vol 187 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Karen Lipkow ◽  
Steven S. Andrews ◽  
Dennis Bray
Keyword(s):  
Escherichia Coli ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Three Dimensional Model ◽  
Large Protein ◽  
E Coli ◽  
Flagellar Motors ◽  
Apparent Diffusion ◽  
A Cell ◽  
Mutant Cells

ABSTRACT We describe the use of a computational model to study the effects of cellular architecture and macromolecular crowding on signal transduction in Escherichia coli chemotaxis. A newly developed program, Smoldyn, allows the movement and interaction of a large number of individual molecules in a structured environment to be simulated (S. S. Andrews and D. Bray, Phys. Biol., in press). With Smoldyn, we constructed a three-dimensional model of an E. coli cell and examined the diffusion of CheYp from the cluster of receptors to the flagellar motors under control conditions and in response to attractant and repellent stimuli. Our simulations agree well with experimental observations of cell swimming responses and are consistent with the diffusive behavior expected in wild-type and mutant cells. The high resolution available to us in the new program allows us to calculate the loci of individual CheYp molecules in a cell and the distribution of their lifetimes under different cellular conditions. We find that the time delay between stimulus and response differs for flagellar motors located at different positions in the cell. We explore different possible locations for the phosphatase CheZ and show conditions under which a gradient of CheYp exists in the cell. The introduction of inert blocks into the cytoplasm, representing impenetrable structures such as the nucleoid and large protein complexes, produces a fall in the apparent diffusion coefficient of CheYp and enhances the differences between motors. These and other results are left as predictions for future experiments.

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