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Biomedical subjects

A C Steven

Publications and source records attributed to A C Steven.

At least 145 records · Page 8Linked to original sources
PubMed1980

Fidelity of structure representation in electron micrographs of negatively stained protein molecules.

We have investigated the fidelity of structure representation in electron micrographs of negatively stained proteins by conducting a systematic evaluation of such micrographs in terms of a known molecular structure, solved by x-ray crystallography. Microcrystals of immunoglobulin G Dob were used as specimens in this comparison between micrograph images, optimized by computer image processing, and reference images derived computationally from the crystal structure. To an effective resolution of 2 nm, we observed a remarkably good correlation between the experimental images and their idealized counterparts, which are unaffected by those factors--electron irradiation and dehydration--that are thought to be primarily responsible for perturbation of protein structure during electron microscopy. Separate structural features resolved in these micrographs do not, in general, correspond to specific components of individual molecules but arise instead from complex superpositions involving several overlapping molecules.

Crystallography↗
PubMed1979

Proteolytic cleavage and structural transformation: their relationship in bacteriophage T4 capsid maturation.

Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp23 monomers excised by phage-specific proteolysis. Cleaved 17- giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.

Coliphages↗
PubMed1978

Paracrystalline arrays of protein-synthesis elongation factor Tu. Comparison with polymerized actin.

Homogeneous protein synthesis elongation factor Tu from Escherichia coli forms aggregates at high concentrations of ammonium sulfate which have a filamentous appearance in the light microscope. Electron microscopy of negatively stained preparations shows that these aggregates are paracrystalline, including three different forms. On the basis of analyses by optical diffraction, this polymorphism can be explained in terms of three different tubular foldings of the same basic two-dimensional surface lattice. This can be compared with that underlying the structure of actin filaments, thus providing a crucial test of the putative relationship between the elongation factor and actin [Rosenbusch, J. P. et al. (1976) J. Supramol. Struct. 5, 391-396]. The differences between the surface lattices, in conjunction with the negative results of sensitive immunochemical tests for possible cross-reactivities between the two proteins, suggest that any such relationship is very remote.

Actins↗
PubMed1977

Ultrastructure of a periodic protein layer in the outer membrane of Escherichia coli.

Matrix protein (36,500 daltons), one of the major polypeptides of the Escherichia coli cell envelope, is arranged in a periodic monolayer which covers the outer surface of the peptidoglycan. Although its association with the peptidoglycan layer is probably tight, the periodic structure of the peptidoglycan. Although its association with the peptidoglycan later is probably tight, the periodic structure is maintained in the absence of peptidoglycan, and is therefore based on strong protein-protein interactions. A detailed analysis of the ultrastructure of the matrix protein array by electron microscopy and image processing of specimens prepared by negative staining or by freeze-drying and shadowing shows that the molecules are arranged according to three fold symmetry on a hexagonal lattice whose repeat is 7.7 nm. The most pronounced feature of the unit cell, which probably contains three molecules of matrix protein, is a triplet of indentations, each approx. 2 nm in diameter, with a center-to-center spacing of 3nm. They are readily penetrated by stain and may represent channels which span the protein monolayer.

Bacterial Proteins↗
PubMed1976

Comparison of the structural and chemical composition of giant T-even phage heads.

A study has been made of the structure of the capsids of T4D giant phage produced from mutants in gene 23 and temperature-sensitive mutants in gene 24, and T4D and T2L giant phage formed by the addition of L-canavanine followed by an Larginine chase in the growth medium. All the giant phage capsids have been shown to be built according to the same geometrical architecture. This consists of a near-hexagonal surface net, lattice constant 129.5 A, folded into a left-handed T = 13 prolate icosahedron elongated along one of its fivefold symmetry axes. Their only apparent difference from wild-type T-even phage capsids is their abnormally elongated tubular part. A comparison of the capsomere morphologies and protein compositions of the giant phage capsids showed that all T4D giants are identical but differ from T2L: The T4D capsomere has a complex (6 + 6 + 1)-type morphology, whereas the T2L has a simple 6-type. T2L phage, however, lack two capsid proteins, "soc" and "hoc", present in T4D. The difference in capsomere morphology can therefore be related to the difference in the protein compositions of these two phage. Possible differences between the initiation and means of length regulation of giant phage heads and the aberrant polyheads are discussed.

Capsid↗
PubMedDate not supplied

The effects of radiation damage on the structure of frozen hydrated HSV-1 capsids.

Radiation damage imposes stringent limits on the information content of electron micrographs of biological specimens. In this study, we have investigated its effects on frozen, hydrated specimens and three-dimensional reconstructions calculated from cryomicrographs using capsids of herpes simplex virus as a model system. Multiple-exposure series of micrographs of both B-capsids (which contain no DNA) and C-capsids (which are fully packaged) were recorded and reconstructions were calculated from the first exposures, corresponding to a cumulative electron dose of 6-7 e-/A2, and from later exposures (25-40 e-/A2). Experimental procedures were standardized to ensure that perceived changes in the micrographs and reconstructions would be attributable to radiation damage alone. The effects of the higher doses in both the micrographs and the reconstructions were expressed as a progressive blurring of the finer details, corresponding to a delocalization of structure in the ice-embedded specimens. The resolutions of the reconstructions were quantified according to a form of the Fourier ring correlation coefficient criterion, according to which the first-exposure reconstructions had resolutions of 30-36 A. The fifth-exposure B-capsid reconstruction had comparable nominal resolution, although it exhibited progressively lower correlations at higher spatial frequencies. Qualitatively similar changes in the series of C-capsid reconstructions were observed although they were more pronounced, presumably because these micrographs had lower contrast and signal-to-noise ratios. We infer that the observed changes in the images and reconstructions and the concomitant loss in contrast in the immediate vicinity of the capsid surface may reflect radiation-induced perturbation of molecular structure and/or the release of peptide fragments. Nevertheless, the observed changes are relatively subtle, at least at the operational resolution of this study; overall, our results support earlier indications (M. F. Schmid et al. J. Struct. Biol. 108, 62-68, 1992) that prospects are quite good for tilt-series reconstructions from cryoelectron micrographs, including six to eight views of the same specimen.

Capsid↗
PubMedDate not supplied

Digital image processing of electron micrographs: the PIC system-III.

The PIC system is an integrated package of image processing software written in Fortran and C. Throughout its 16 years of continuous development, PIC has been designed for the processing of electron micrographs with emphasis on the particular requirements for structural analysis of biological macromolecules. PIC has been implemented on successive generations of Digital Equipment Corporation dedicated minicomputers and workstations. The latest version, PIC-III, runs on Alpha workstations and represents a substantial upgrading in functionality compared with PIC-II, the VAX version previously described (B.L. Trus et al. (1992), Scanning Microsc. Suppl. 6, 441-451). PIC now possesses an X-windows menu-driven graphical user interface that many be utilized from both local or remote X-window terminals or workstations. Other new features include dynamic memory allocation; an open systems approach to interfacing with other image processing software packages; interface options with visualization software; and programs to reconstruct three-dimensional density maps of both helical filaments and free-standing particles with and without symmetry.

Computer Simulation↗
PubMedDate not supplied

Visualization of three-dimensional density maps reconstructed from cryoelectron micrographs of viral capsids.

Full evaluation of three-dimensional density maps calculated from cryoelectron micrographs of complex supramolecular structures requires that the maps be sifted by a variety of complementary visualization techniques. We present here a primer for a number of such techniques in current widespread use, including surface rendering; serial sections; simulated motion; and real-time manipulation of tiled surfaces displayed on an advanced workstation. The principles on which these techniques operate are briefly reviewed, as are their advantages and limitations, with emphasis on the requirements for visual representation of viral capsid structures. These methods are illustrated in application to a density map of herpes simplex virus type 1 (HSV-1) capsid at 24 A resolution, which reveals more detailed information than heretofore concerning the inner surface of the icosahedral capsid shell and the 150-A-long channels that pass through each of the 162 capsomers.

Animals↗
PubMedDate not supplied

Three-dimensional structure of Bordetella pertussis fimbriae.

We describe the helical structure of Bordetella pertussis fimbriae of serotype 3/6 as determined to a resolution of approximately 2.5 nm by three-dimensional reconstruction of negatively stained electron micrographs. The fimbria has a distinctly polar structure whose axial repeat of 13 nm contains five copies of the fim3 gene product (22 kDa) in two complete turns. These subunits are connected by interactions along the fimbrial backbone which, unlike other classes of bacterial fimbriae, has no axial channel. Its outer diameter is approximately 5.7 nm, and the most pronounced feature is a radially protruding domain that gives the fimbria its characteristic serrated appearance. Serotype 2 fimbriae, composed of the fim2 subunit which is 60% homologous with fim3, have essentially the same quaternary structure. These observations are discussed in relation to fimbrial phase variation and structure-based classification of fimbriae/pili.

Antigens, Bacterial↗
PubMedDate not supplied

A strategy for determining the orientations of refractory particles for reconstruction from cryo-electron micrographs with particular reference to round, smooth-surfaced, icosahedral viruses.

Cryo-electron microscopy and three-dimensional image reconstruction are powerful tools for analyzing icosahedral virus capsids at resolutions that now extend below 1 nm. However, the validity of such density maps depends critically on correct identification of the viewing geometry of each particle in the data set. In some cases-for example, round capsids with low surface relief-it is difficult to identify orientations by conventional application of the two most widely used approaches-"common lines" and model-based iterative refinement. We describe here a strategy for determining the orientations of such refractory specimens. The key step is to determine reliable orientations for a base set of particles. For each particle, a list of candidate orientations is generated by common lines: correct orientations are then identified by computing a single-particle reconstruction for each candidate and then systematically matching their reprojections with the original images by visual criteria and cross-correlation analysis. This base set yields a first-generation reconstruction that is fed into the model-based procedure. This strategy has led to the structural determination of two viruses that, in our hands, resisted solution by other means.

Algorithms↗
PubMedDate not supplied

Normalization procedures and factorial representations for classification of correlation-aligned images: a comparative study.

We have addressed the problem of optimizing procedures of multivariate statistical analysis (MSA) for identifying homogeneous sets of electron micrographs of biological macromolecules, with a view to averaging over consistent sets of images. Using pre-aligned images of negatively stained protein molecules - known a priori to fall into two subtly different classes - we compared how the capacity to discriminate between them was affected by the normalization procedure used, and by the choice of factorial representation. Specifically, these images were analyzed both after being scaled according to constant minimum and maximum (CMM) values, and after imposing constant values of image mean and variance (CMV). The factorial representations compared were correspondence analysis (CA) and the principal components (PC) formalism. When used with PC, CMM normalization was found to give rise to spurious inter-image fluctuations that were more pronounced than the genuine difference between the two kinds of images; even with CA, CMV proved to be a more satisfactory method of normalization. When CMV was used with CA or PC, both factorial representations yielded qualitatively similar results, although according to a quantitative measure of inter-set discrimination, the performance of PC was slightly superior. Even in the best case, however, the two classes of images - as mapped in factorial space - were not fully resolved. The implications of this observation are discussed with regard to potential ambiguities of image classification in practice.

Classification↗
PubMedDate not supplied

The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision.

This note describes a practical improvement in the computational efficiency of the spectral signal-to-noise ratio (SSNR) resolution criterion for correlation-averaged images. The total set of N images is randomly partitioned into ng subsets, each subset is separately averaged, and a reduced form of the SSNR is computed from these average images. In general, larger values of ng achieve lower statistical uncertainty, while smaller values of ng are computationally more expedient. It is shown that, for negatively stained data, a judicious compromise is achieved with 10 less than or equal to ng less than or equal to 20, regardless of how large N may be.

Microscopy, Interference↗