scholarly journals Structure and function at the lipid–protein interface of a pentameric ligand-gated ion channel

2021 ◽  
Vol 118 (23) ◽  
pp. e2100164118
Author(s):  
Pramod Kumar ◽  
Gisela D. Cymes ◽  
Claudio Grosman
Keyword(s):  
Ion Channel ◽  
Lipid Bilayers ◽  
Secretory Pathway ◽  
Three Dimensional ◽  
E Coli ◽  
Density Maps ◽  
Structural Relevance ◽  
Bound Lipids ◽  
Dimensional Reconstruction ◽  
And Function

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli’s inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein–bulk-membrane interface. One of them was consistent with a “regular” diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, “resting” conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

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.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

Predicting spatial activity patterns in a neural map from a probabilistic atlas of 3-D reconstructed neurons

1995 ◽  
Vol 53 ◽  
pp. 812-813
Author(s):  
G. Jacobs ◽  
F. Theunissen
Keyword(s):  
Activity Patterns ◽  
Three Dimensional ◽  
Sensory System ◽  
Neural System ◽  
Probabilistic Atlas ◽  
Uniform Sample ◽  
Dimensional Reconstruction ◽  
The Time Domain ◽  
And Function ◽  
Visualization Techniques

In order to understand how the algorithms underlying neural computation are implemented within any neural system, it is necessary to understand details of the anatomy, physiology and global organization of the neurons from which the system is constructed. Information is represented in neural systems by patterns of activity that vary in both their spatial extent and in the time domain. One of the great challenges to microscopists is to devise methods for imaging these patterns of activity and to correlate them with the underlying neuroanatomy and physiology. We have addressed this problem by using a combination of three dimensional reconstruction techniques, quantitative analysis and computer visualization techniques to build a probabilistic atlas of a neural map in an insect sensory system. The principal goal of this study was to derive a quantitative representation of the map, based on a uniform sample of afferents that was of sufficient size to allow statistically meaningful analyses of the relationships between structure and function.

Three-Dimensional Reconstruction of Two Forms of the Chaperonin GRO EL

1990 ◽  
Vol 48 (1) ◽  
pp. 258-259
Author(s):  
J.L. Carrascosa ◽  
G. Abella ◽  
S. Marco ◽  
M. Muyal ◽  
J.M. Carazo
Keyword(s):  
Three Dimensional ◽  
The Other ◽  
Two Dimensional ◽  
Series Method ◽  
Three Dimensional Reconstruction ◽  
Structural Components ◽  
E Coli ◽  
Dimensional Reconstruction ◽  
Morphogenetic Factors ◽  
Tilt Series

Chaperonins are a class of proteins characterized by their role as morphogenetic factors. They trantsiently interact with the structural components of certain biological aggregates (viruses, enzymes etc), promoting their correct folding, assembly and, eventually transport. The groEL factor from E. coli is a conspicuous member of the chaperonins, as it promotes the assembly and morphogenesis of bacterial oligomers and/viral structures.We have studied groEL-like factors from two different bacteria:E. coli and B.subtilis. These factors share common morphological features , showing two different views: one is 6-fold, while the other shows 7 morphological units. There is also a correlation between the presence of a dominant 6-fold view and the fact of both bacteria been grown at low temperature (32°C), while the 7-fold is the main view at higher temperatures (42°C). As the two-dimensional projections of groEL were difficult to interprete, we studied their three-dimensional reconstruction by the random conical tilt series method from negatively stained particles.

scholarly journals Conserved Structure and Function in the Granulysin and NK-Lysin Peptide Family

2005 ◽  
Vol 73 (10) ◽  
pp. 6332-6339 ◽  
Author(s):  
Charlotte M. A. Linde ◽  
Susanna Grundström ◽  
Erik Nordling ◽  
Essam Refai ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Antimycobacterial Activity ◽  
Structure And Function ◽  
Loop Structure ◽  
E Coli ◽  
Short Loop ◽  
Peptide Family ◽  
And Function ◽  
Related Proteins

ABSTRACT Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variable Escherichia coli activities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity against E. coli to a larger extent than that against Mycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria.

scholarly journals Three dimensional reconstruction to visualize atrial fibrillation activation patterns on curved atrial geometry

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249873
Author(s):  
Ricardo Abad ◽  
Orvil Collart ◽  
Prasanth Ganesan ◽  
A. J. Rogers ◽  
Mahmood I. Alhusseini ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Three Dimensional ◽  
Spiral Waves ◽  
Patient Specific ◽  
Activation Patterns ◽  
Density Maps ◽  
Median Correlation ◽  
Geometric Curvature ◽  
Dimensional Reconstruction ◽  
Phase Singularities

Background The rotational activation created by spiral waves may be a mechanism for atrial fibrillation (AF), yet it is unclear how activation patterns obtained from endocardial baskets are influenced by the 3D geometric curvature of the atrium or ‘unfolding’ into 2D maps. We develop algorithms that can visualize spiral waves and their tip locations on curved atrial geometries. We use these algorithms to quantify differences in AF maps and spiral tip locations between 3D basket reconstructions, projection onto 3D anatomical shells and unfolded 2D surfaces. Methods We tested our algorithms in N = 20 patients in whom AF was recorded from 64-pole baskets (Abbott, CA). Phase maps were generated by non-proprietary software to identify the tips of spiral waves, indicated by phase singularities. The number and density of spiral tips were compared in patient-specific 3D shells constructed from the basket, as well as 3D maps from clinical electroanatomic mapping systems and 2D maps. Results Patients (59.4±12.7 yrs, 60% M) showed 1.7±0.8 phase singularities/patient, in whom ablation terminated AF in 11/20 patients (55%). There was no difference in the location of phase singularities, between 3D curved surfaces and 2D unfolded surfaces, with a median correlation coefficient between phase singularity density maps of 0.985 (0.978–0.990). No significant impact was noted by phase singularities location in more curved regions or relative to the basket location (p>0.1). Conclusions AF maps and phase singularities mapped by endocardial baskets are qualitatively and quantitatively similar whether calculated by 3D phase maps on patient-specific curved atrial geometries or in 2D. Phase maps on patient-specific geometries may be easier to interpret relative to critical structures for ablation planning.

Proteolytic Control of Bacteriophage HK97 Capsid Maturation.

1998 ◽  
Vol 4 (S2) ◽  
pp. 984-985
Author(s):  
Robert L. Duda ◽  
James F. Conway ◽  
Naiqian Cheng ◽  
Alasdair C. Steven ◽  
Roger W. Hendrix
Keyword(s):  
Conformational Changes ◽  
Three Dimensional ◽  
Temperate Bacteriophage ◽  
Covalent Bonds ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dimensional Reconstruction ◽  
Capsid Maturation ◽  
Icosahedral Capsid ◽  
Icosahedral Particle

HK97 is a tailed temperate bacteriophage of E. coli that builds an icosahedral capsid using steps that include regulated assembly, proteolysis, radical conformational changes and the formation of novel covalent bonds (Fig. 1). This pathway is being exploited as a model system to explore how the formation of multiprotein complexes can be regulated by each of these mechanisms. We have identified and purified at least four intermediates (Prohead I, Prohead II, Head I and Head II) and examined them by cryo-electron microscopy and three dimensional reconstruction procedures (Fig. 2). Comparison of particle reconstructions at resolution of about 25 - 30 A have lead to major insights into the causes and purposes of the regulated changes that we have also characterized biochemically and genetically.Prohead I consists of 420 copies of the 42 kDa gp5 capsid protein arranged as 72 blister-shaped morphological capsomers in a thick walled hollow T=7 icosahedral particle with a diameter of -470 Å.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document