Three-Dimensional Image Reconstruction of the 50S Subunit from Escherichia Coli Ribosomes Lacking 5S-rRNA

1990 ◽  
Vol 48 (1) ◽  
pp. 284-285
Author(s):  
Michael Radermacher ◽  
Volker Nowotny ◽  
Robert Grassucci ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
5S Rrna ◽  
Scattering Data ◽  
23S Rrna ◽  
Reconstruction Technique ◽  
Electron Dose ◽  
E Coli ◽  
Phenol Extraction ◽  
Negative Stain

In earlier studies the structure of the 50S ribosomal subunit from E. coli has been determined from electron micrographs, using the single exposure random conical reconstruction technique. For the understanding of the function of ribosomes the single proteins and ribosomal RNAs need to be located within the ribosome structure. For localization of most proteins immunoelectron microscopy and neutron scattering data are available. The current study’s goal is the localization of the 5S rRNA from a comparison of the structures of complete 50S subunits with that of subunits reconstituted omitting the 5S rRNA.By Phenol extraction of purified 50S subunits the rRNA fraction was separated form the total protein fraction (TP50). This rRNA fraction was separated into 23S rRNA and 5S rRNA via HPLC on a DEAE-column. The total reconstitution of 23S rRNA and the equivalent amount of TP50 resulted in particles lacking the 5S rRNA. For electron microscopy the subunits were prepared in a negative stain sandwich preparation.Twenty tilt pairs at 50° tilt and 0° were recorded, with an electron dose of approximately 10el/A2 and a magnification of 49,000. A total of 983 particles were selected from these data. The 0° images were aligned using the procedure described in.

The Three-dimensional structure of frozen-hydrated nudaurelia capensis β virus

1987 ◽  
Vol 45 ◽  
pp. 650-651
Author(s):  
N. H. Olson ◽  
T. S. Baker ◽  
Wu Bo Mu ◽  
J. E. Johnson ◽  
D. A. Hendry
Keyword(s):  
South African ◽  
Rna Virus ◽  
Carbon Film ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Electron Dose ◽  
Total Electron ◽  
Three Dimensional Structure ◽  
Negative Stain ◽  
Dimensional Reconstruction

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

1987 ◽  
Vol 45 ◽  
pp. 936-937
Author(s):  
Neng-Yu Zhang ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
Tom Obrig ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Uranyl Acetate ◽  
Reconstruction Method ◽  
Single Exposure ◽  
Electron Dose ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

scholarly journals A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

1973 ◽  
Vol 133 (4) ◽  
pp. 739-747 ◽  
Author(s):  
A. Robinson ◽  
J. Sykes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Core Particle ◽  
Protein Loss ◽  
E Coli ◽  
Rrna Species ◽  
Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

Negative staining versus rotational evaporation

1983 ◽  
Vol 41 ◽  
pp. 598-599
Author(s):  
H. Schmiady ◽  
C. Kreuzfeldt ◽  
E. Reuber ◽  
B. Tesche
Keyword(s):  
Test Specimen ◽  
Staining Method ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Negative Staining ◽  
Positive Staining ◽  
Negative Stain ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Tilt Series

This comparative study demonstrates the salient differences between the two most common methods of contrasting subcellular particles, using the 40S ribosomal subunit from saccharomyces cerevisiae as test specimen because its socalled beak-like L (left) and R (right) particle projections can be unequivocally distinguished.The negative staining method produces images which are not artifact-free because it is a combination of fixation and chemical staining and, therefore, not neutral towards the object. For three-dimensional reconstruction it is unsatisfactory because:(1) The affinity of the support film to the negative stain solution differs so greatly that sometimes undesired positive staining effects occur.(2) The rendition of the structure depends on the embedding--that is, on the level of the negative stain; fluctuations in the level lead to a loss and/or change of the structure, and thus to difficulties in interpreting the stain distribution, especially when reconstruction of the object by means of tilt series is planned.

scholarly journals The rlmB Gene Is Essential for Formation of Gm2251 in 23S rRNA but Not for Ribosome Maturation inEscherichia coli

2001 ◽  
Vol 183 (23) ◽  
pp. 6957-6960 ◽  
Author(s):  
J. Mattias Lövgren ◽  
P. Mikael Wikström
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Cell Mass ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Ribosome Assembly ◽  
Large Ribosomal Subunit ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
E Coli

ABSTRACT In Saccharomyces cerevisiae, the rRNA Gm2270 methyltransferase, Pet56p, has an essential role in the maturation of the mitochondrial large ribosomal subunit that is independent of its methyltransferase activity. Here we show that the proposedEscherichia coli ortholog, RlmB (formerly YjfH), indeed is essential for the formation of Gm in position 2251 of 23S rRNA. However, a ΔrlmB mutant did not show any ribosome assembly defects and was not outgrown by a wild-type strain even after 120 cell mass doublings. Thus, RlmB has no important role in ribosome assembly or function in E. coli.

scholarly journals cfrB, cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

2019 ◽  
Author(s):  
Vanja Stojković ◽  
María Fernanda Ulate ◽  
Fanny Hidalgo-Villeda ◽  
Emmanuel Aguilar ◽  
Camilo Monge-Cascante ◽  
...  
Keyword(s):  
Latin America ◽  
Ribosomal Proteins ◽  
Functional Characterization ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Cross Resistance ◽  
Protection Factor ◽  
E Coli

ABSTRACTCfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

Three-dimensional reconstruction of the 30S ribosomal subunit from randomly oriented particles

1983 ◽  
Vol 41 ◽  
pp. 758-759
Author(s):  
A. Verschoor ◽  
J. Frank ◽  
M. Radermacher ◽  
T. Wagenknecht ◽  
M. Boublik
Keyword(s):  
Correspondence Analysis ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Two Dimensional ◽  
Mapping Algorithm ◽  
E Coli ◽  
Wide Range ◽  
Dimensional Reconstruction ◽  
Significant Factors

The small (30S) subunit of prokaryotic ribosomes can assume any of a wide range of tilt positions on the specimen support. Correspondence analysis should make it possible to order views appearing in the electron micrograph according to the angle of tilt.231 individual windowed images from two micrographs showing negatively stained 30S subunits from E. coli ribosomes were subjected to multireference alignment. Correspondence analysis yielded six morphologically significant factors of variance. The second of these related to variations in stain concentrations, which are irrelevant at the level of gross morphology. The coordinates for each image in five-dimensional space (relating to factors 1,3,4,5, and 6) were subjected to a nonlinear mapping algorithm, which calculated an optimal two-dimensional map.The resulting distribution (Fig 1) consisted of two clusters, one of rightfacing, the other of left-facing views. Subaverages along the outer margin of the cluster on the left showed the particle in a range of typical views.

Effect of point mutations at position 89 of the E. coli 5S rRNA on the assembly and activity of the large ribosomal subunit

FEBS Letters ◽  
1998 ◽  
Vol 421 (3) ◽  
pp. 249-251 ◽  
Author(s):  
Maria I Zvereva ◽  
Olga V Shpanchenko ◽  
Olga A Dontsova ◽  
Knud H Nierhaus ◽  
Alexey A Bogdanov
Keyword(s):  
Point Mutations ◽  
Ribosomal Subunit ◽  
5S Rrna ◽  
Large Ribosomal Subunit ◽  
E Coli

3D reconstruction of the 50s ribosomal subunit of escherichia coli

1986 ◽  
Vol 44 ◽  
pp. 40-41
Author(s):  
M. Radermacher ◽  
T. Wagenknecht ◽  
A. Verschoor ◽  
J. Frank
Keyword(s):  
3D Reconstruction ◽  
3D Structure ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Main Body ◽  
Large Ribosomal Subunit ◽  
E Coli ◽  
Reconstruction Scheme ◽  
Particle Preparation ◽  
Electron Microscopical

The three-dimensional (3D) structure of the large ribosomal subunit from E. coli was determined from micrographs of a negatively stained 50S particle preparation using our new reconstruction scheme. The 50S subunit occurs in electron microscopical preparations mainly in the crown-view orientation with the interface side of the main body situated parallel to the specimen plane, but in random in plane orientations. An image of such a specimen tilted by a large tilt angle, which inherently contains a conical tilt series of the particle, was used to calculate a 3D reconstruction.

scholarly journals A Negative Stain for Electron Microscopic Tomography

2012 ◽  
Vol 18 (2) ◽  
pp. 331-335 ◽  
Author(s):  
Andrea Fera ◽  
Jane E. Farrington ◽  
Joshua Zimmerberg ◽  
Thomas S. Reese
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Three Dimensional ◽  
Surface Proteins ◽  
Negative Staining ◽  
Electron Microscopic ◽  
Electron Dose ◽  
Tomographic Images ◽  
Negative Stain ◽  
Viral Surface

AbstractWhile negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

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