Angular Calibration of Ribosomal Subuntts in Factor Space

1990 ◽  
Vol 48 (1) ◽  
pp. 462-463
Author(s):  
Minakhi Pujari ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Carbon Layer ◽  
Uranyl Acetate ◽  
Particle Analysis ◽  
Factor Space ◽  
Multivariate Statistical ◽  
Ribosomal Subunits ◽  
Layer Method ◽  
Dimensional Reconstruction ◽  
Number Of Particles

In single-particle analysis of macromolecule images with the electron microscope, variations of projections are often observed that can be attributed to the changes of the particle’s orientation on the specimen grid (“rocking”). In the multivariate statistical analysis (MSA) of such projections, a single factor is often found that expresses a large portion of these variations. Successful angle calibration of this “rocking factor” would mean that correct angles can be assigned to a large number of particles, thus facilitating three-dimensional reconstruction.In a study to explore angle calibration in factor space, we used 40S ribosomal subunits, which are known to rock around an axis approximately coincident with their long axis. We analyzed micrographs of a field of these particles, taken with 20° tilt and without tilt, using the standard methods of alignment and MSA. The specimen was prepared with the double carbon-layer method, using uranyl acetate for negative staining. In the MSA analysis, the untilted-particle projections were used as active, the tilted-particle projections as inactive objects. Upon tilting, those particles whose rocking axes are parallel to the tilt axis will change their appearance in the same way as under the influence of rocking. Therefore, each vector, in factor space, joining a tilted and untilted projection of the same particle can be regarded as a local 20-degree calibration bar.

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.

Structure of ribosomes from a thermophilic bacterium

1989 ◽  
Vol 47 ◽  
pp. 1026-1027
Author(s):  
Janet Dickie ◽  
Karen Kyle ◽  
Derrick Flannigan ◽  
George Harauz
Keyword(s):  
Hot Springs ◽  
Thermophilic Bacteria ◽  
Structural Information ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Thermophilic Bacterium ◽  
Single Particles ◽  
Multivariate Statistical ◽  
Ribosomal Subunits ◽  
Dimensional Reconstruction

The most direct and informative probe of ribosome structure is electron microscopy (EM), of negatively stained and antibody-labelled specimens. Ribosomes from Escherichia coli have been the most intensively studied, although those from other prokaryotes and eukaryotes have also been imaged [e.g., 3, 4, 5],The amount of structural information obtained from electron images can be significantly increased using multivariate statistical analysis and classification techniques [e.g., 6]. Noisy electron micrographs of single particles are sorted according to their principal features, and average images formed with an enhanced signal-to-noise ratio and better reproducible resolution. The class averages represent projections from which a three-dimensional reconstruction can be computed.Thermus aguaticus is a species of extremely thermophilic bacteria isolated from environments such as hot springs. This species possesses macromolecular enzyme complexes with a great thermostability which must be reflected somehow instructural differences. In this study, we investigate the structures of ribosomes and ribosomal subunits from this bacterium.

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.

scholarly journals Three Dimensional Reconstruction of CFTR Chloride Channel Using Single Particle Analysis

2009 ◽  
Vol 96 (3) ◽  
pp. 468a
Author(s):  
Kazuhiro Mio ◽  
Toshihiko Ogura ◽  
Muneyo Mio ◽  
Hiroyasu Shimizu ◽  
Tzyh-Chang Hwang ◽  
...  
Keyword(s):  
Single Particle ◽  
Chloride Channel ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Three Dimensional Reconstruction ◽  
Dimensional Reconstruction

Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)

2012 ◽  
Vol 449 (2) ◽  
pp. 365-371 ◽  
Author(s):  
Ziguo Zhang ◽  
Jing Yang ◽  
Eric H. Kong ◽  
William C. H. Chao ◽  
Edward P. Morris ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Recombinant Expression ◽  
Three Dimensional ◽  
Homology Model ◽  
Particle Analysis ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Synthetic Lethal ◽  
Dimensional Reconstruction ◽  
A Subunit

Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.

scholarly journals High-resolution cryo-EM proteasome structures in drug development

2017 ◽  
Vol 73 (6) ◽  
pp. 522-533 ◽  
Author(s):  
Edward P. Morris ◽  
Paula C. A. da Fonseca
Keyword(s):  
Structure Determination ◽  
Protein Structures ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Particle Analysis ◽  
20S Proteasome ◽  
Ligand Specificity ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Dimensional Reconstruction

With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein–ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein–ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of thePlasmodium falciparum20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human andP. falciparum20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of theicr3dsoftware used for three-dimensional reconstruction.

Challenges of three-dimensional reconstruction of ribonucleoprotein complexes from electron spectroscopic images - Reconstructing ribosomal RNA

1996 ◽  
Vol 54 ◽  
pp. 52-53
Author(s):  
D.R. Beniac ◽  
G.J. Czarnota ◽  
T.A. Bartlett ◽  
F.P. Ottensmeyer ◽  
G. Harauz
Keyword(s):  
Ribosomal Rna ◽  
Correlation Functions ◽  
Three Dimensional ◽  
Three Dimensional Reconstruction ◽  
Mutual Correlation ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Ribonucleoprotein Complexes ◽  
Dimensional Reconstruction

Transmission electron microscopy has been dominant in structural studies of the ribosome and its constituent ribonucleic acids and proteins. Ribosomal RNA (rRNA) has central importance in the architecture of this complex and in protein synthesis. Our work has entailed using electron spectroscopic imaging (ESI) to probe the tertiary structure of rRNA in situ in a prokaryote (Escherichia coli) and in a eukaryote (Thermomyces lanuginosus). ESI uses only electrons which have lost a specific amount of energy due to specific inner-shell ionisation interactions with the specimen to form an elemental map. In nucleoprotein complexes, a map of the phosphorus distribution represents primarily a projection of the phosphate backbone of the nucleic acid component. The visualisation of rRNA in situ in the intact ribosomal subunit by ESI was demonstrated almost a decade ago to be feasible. Our work on quantitative image analysis of ES images of E. coli and Th. lanuginosus ribosomal subunits has presented unique challenges and has resulted in new algorithmic developments generally applicable to such images. These innovations include a singular pretreatment procedure, the use of mutual correlation functions rather than cross correlation functions to reduce the effect of low spatial frequency components, and angular determination using iterative quaternion-assisted angular reconstitution to compute a three-dimensional reconstruction. These investigations have produced direct information regarding ribosomal rRNA localisation in the ribosomal subunits of E. coli and Th. lanuginosus, and the position of non-conserved sequences.

scholarly journals Probing Ribosomal RNA By Electron Spectroscopic Imaging and Three-Dimensional Reconstruction

1997 ◽  
Vol 5 (1) ◽  
pp. 10-11
Author(s):  
Daniel R. Beniac ◽  
Gregory J. Czarnota ◽  
Brenda L. Rutherford ◽  
F. Peter Ottensmeyer ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Elemental Distribution ◽  
Two Dimensional ◽  
Electron Spectroscopic Imaging ◽  
Ribosomal Subunits ◽  
Microanalytical Technique ◽  
Dimensional Reconstruction

The ribosome is the protein synthetic machinery in the cell. Knowledge of the structures of ribosomal RNA (rRNA) macromolecules in situ is essential to understanding their roles in ribosome mediated protein synthesis. We are using a microanalytical technique that identifies and maps elements directly, electron spectroscopic imaging, to determine the rRNA phosphorus distributions within Escherichia coli ribosomal subunits, and to combine the two-dimensional maps into a three-dimensional elemental distribution by iterative quaternion-assisted angular reconstitution of ribosomal particles at random orientations.

Sign in / Sign up

Export Citation Format

Share Document