Introduction to the computer image processing of electron micrographs of two‐dimensionally ordered biological structures

Stewart, Murray

doi:10.1002/jemt.1060090403

Cited by 47 publications

(25 citation statements)

References 59 publications

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“…CCA and lattice unbending had to be used because shortand long-range orders of the crystalline S layer were generally poor (38). A least-squares fit of an ideal lattice to the found correlation maxima gave a base vector length of 16.0 + 0.7 nm for the hexagonal lattice.…”

Section: Methodsmentioning

confidence: 99%

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense

Pum¹,

Messner²,

Sleytr³

1991

J Bacteriol

119

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Thin sections, freeze-etched, and negatively stained preparations of Methanocorpusculum sinense cells reveal a highly lobed cell structure with a hexagonally arranged surface layer (S layer). Digital image processing of negatively stained envelope fragments show that the S layer forms a porous but strongly interconnected network. Since the S layer is the exclusive cell envelope component outside the cytoplasmic membrane it must have a cell shape determining and maintaining function. Although lattice faults such as disclinations and dislocations are a geometrical necessity on the surface of a closed protein crystal, our data indicate that they also play important roles as sites for the incorporation of new morphological units, in the formation of the lobed cell structure, and in the cell division process. In freeze-etched preparations of intact cells numerous positive and negative 600 wedge disclinations can be detected which form pentagons and heptagons in the hexagonal array. Complementary pairs of pentagons and heptagons are the termination points of edge dislocations. They can be expected to function both as sites for incorporation of new morphological units into the lattice and as initiation points for the cell division process. The latter is determined by the ratio between the increase of protoplast volume and the increase in actual S-layer surface area during cell growth. We postulate that this mode of cell fission represents a common feature in lobed archaebacteria which possess an S layer as the exclusive wall component. Two-dimensional crystalline surface layers, termed S layers (36), have been observed as the outermost cell envelope component in many strains of walled eubacteria and represent an almost universal feature of archaebacterial cell envelopes (for recent compilations, see references 24, 33, and 36). Most of the presently known S layers are composed of a single protein (in some cases, a glycoprotein) species with molecular weights ranging from 40,000 to 200,000. S layers cover the bacterial cell completely and are organized in the form of crystalline lattices exhibiting oblique (p2), trigonal (p3), square (p4), or hexagonal (p6) symmetry. Isolated S-layer subunits have shown the inherent ability to self-assemble into two-dimensional arrays, either in the presence or absence of surfaces suitable for adhesion (for reviews, see references 31 and 35). These self-assembly studies have shown that all the information required for generating the two-dimensional crystalline arrays is entirely contained in the individual protomeric units (30). Considering S layers as porous crystalline arrays covering the cell surface completely, they have the potential to function as (i) protective coats, molecular sieves, and molecule and ion traps, (ii) structures involved in cell adhesion and surface recognition, and (iii) frameworks which determine and maintain cell shape (for reviews see references 1, 2, 21, 24, 34, and 37).The present study deals with the dynamic process of assembly of the S layer of the archaeb...

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Section: Methodsmentioning

confidence: 99%

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense

Pum¹,

Messner²,

Sleytr³

1991

J Bacteriol

119

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“…To extract the periodic information and determine the symmetry of the lattice in projection, the electron micrographs were filtered to improve the signal to noise ratio (48). This procedure was implemented by computing the Fourier transform of the image, multiplying the transform by a set of Gaussian functions (one around each of the reciprocal lattice points), and then computing a back Fourier transform to obtain the filtered image.…”

Section: Experimental Prodceduresmentioning

confidence: 99%

Electron Microscopy and X-ray Diffraction Studies of Lotus tetragonolobus A Isolectin Cross-linked with a Divalent Lewisx Oligosaccharide, an Oncofetal Antigen

Cheng¹,

Bullitt²,

Bhattacharyya³

et al. 1998

Journal of Biological Chemistry

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The interactions of lectins with multivalent carbohydrates often leads to the formation of highly ordered cross-linked lattices that are amenable to structural studies. A particularly well ordered, two-dimensional lattice is formed from fucose-specific isolectin A from Lotus tetragonolobus cross-linked with difucosyllacto-N-neohexaose, an oligosaccharide possessing the Lewis x determinant, which is an oncofetal antigen. A combination of electron microscopy, x-ray diffraction, simulation of electron micrographs, and molecular model building was used to determine the relative positions of the tetrameric lectin and bivalent carbohydrate within the lattice. X-ray diffraction from unoriented pellets was used to determine the lattice dimensions and analysis of electron micrographs was used to determine the lattice symmetry. Molecular models of the lattice were constructed based on the known structure of the jack bean lectin concanavalin A and the high degree of sequence homology between the two lectins. Using the symmetry and dimensions of the lattice and its appearance in filtered electron micrographs, molecular models were used to determine the orientation of the lectin in the lattice, and to define the range of lectin-oligosaccharide interactions consistent with the structural data. The present study provides the first description of a highly ordered, two-dimensional, cross-linked lattice between a tetravalent lectin and a bivalent carbohydrate.The carbohydrate moieties of glycoproteins and glycolipids have been shown to be involved in a variety of biological recognition processes including cell-cell and cell-substratum interactions, immunity, apoptosis, and metastasis of tumor cells (1-5). The composition and structures of the oligosaccharides correlate with cell differentiation and transformation (cf. Ref. 6). For example, the expression of oligosaccharides possessing specific Lewis blood group antigenic determinants is developmentally regulated and altered as a result of differentiation and oncogenic transformation (7). Some antigens such as the (4) and metastasis (9, 10).The biological signal transduction properties of lectins appear to be due to their ability to bind and cross-link specific glycoprotein and glycolipid receptors on cells. For example, lectin-induced cross-linking of receptors often leads to mitogenic responses in cells (11), in the arrest of bulk transport in ganglion cell axons (12), in the induction of mating reactions in fungi (13), in the molecular sorting of glycoproteins in the secretory pathways of cells (14), and in the apoptosis of activated human T-cells (4). Furthermore, lectin-induced crosslinking of transmembrane glycoproteins leads to changes in their interactions with cytoskeletal proteins and concomitant alterations in the mobility and aggregation of other surface receptors (15,16).Studies have shown that lectins undergo two general types of cross-linking interactions with multivalent carbohydrates, designated type 1 and type 2 complexes (17). In a type 1 complex, binding betwe...

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“…If the protein is Image processing is a powerful method for enhancing stable in detergent, the increased purity of the compo-the resolution and clarity of images of two-dimensional nents in the in vitro method will more likely lead to crystals of biological macromolecules recorded by higher resolution crystals. transmission electron microscopy (for reviews see Amos et al, 1985;Moody, 1990;Stewart, 1988). In order to minimize beam damage, minimal electron dose techniques are used (Williams and Fisher, 19701, and the recorded images are consequently noisy.…”

Section: Resultsmentioning

confidence: 99%

Electron microscopic image analysis of cardiac gap junction membrane crystals

Yeager

1995

Microscopy Res & Technique

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Cardiac gap junctions play an important functional role in the myocardium by electrically coupling adjacent cells, thereby providing a low resistance pathway for cell-to-cell propagation of the action potential. Two-dimensional crystallization of biochemically isolated rat ventricular gap junctions has been accomplished by an in situ method in which membrane suspensions are sequentially dialyzed against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are partially extracted without solubilizing the protein, and the increased protein concentration facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Projection density maps show that the connexons in cardiac gap junctions are formed by a hexameric cluster of alpha 1 connexin subunits. Protease cleavage of alpha 1 connexin from 43 to 30 kDa releases approximately 13kDa from the carboxy-tail, and the projection density maps are not significantly altered. Uranyl acetate stain penetrates the ion channel, whereas phosphotungstic acid is preferentially deposited over the lipid regions. This differential staining can be used to selectively probe the central channel of the connexon and the interface between the connexon and the lipid. The hexameric design of alpha 1 connexons appears to be a recurring quaternary motif for the multigene family of gap junction proteins.

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Introduction to the computer image processing of electron micrographs of two‐dimensionally ordered biological structures

Cited by 47 publications

References 59 publications

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense

Electron Microscopy and X-ray Diffraction Studies of Lotus tetragonolobus A Isolectin Cross-linked with a Divalent Lewisx Oligosaccharide, an Oncofetal Antigen

Electron microscopic image analysis of cardiac gap junction membrane crystals

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