Microfibrillar structure and packing of collagen: hydrophobic interactions.
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Amino Acid Sequence↗
Biomedical subjects
B L Trus
Publications and source records attributed to B L Trus.
Tuna cytochrome c at 2.0 A resolution. I. Ferricytochrome structure analysis.
The crystal structure of oxidized cytochrome c from tuna hearts has been solved by x-ray diffraction to a resolution of 2.0 A, using four isomorphous heavy atom derivatives. The crystals, space group P43, have 2 independent cytochrome molecules in the asymmetric repeating unit. No significant difference is seen between these 2 molecules, aside from conformations of a few surface side chains. The molecular folding observed is essentially that reported for tuna ferrocytochrome c. In particular, the ring of phenylalanine 83 lies against the heme group and closes the heme crevice, and is not swung out into the surroundings as had been believed from the 2.8 A horse ferricytochrome c structure.
Animals↗
Tuna cytochrome c at 2.0 A resolution. II. Ferrocytochrome structure analysis.
The x-ray crystal structure analysis of tuna ferrocytochrome c has been extended from 2.45 to 2.0 A resolution. The overall folding is unchanged and is the same as has been reported for tuna ferricytochrome c (Swanson R., Trus, B.L., Mandel, N., Mandel, G., Kallai, O.B., and Dickerson, R.E. (1977) J. Biol. Chem. 252, 759-755). No significant structural differences are observed between oxidation states. Difference map studies using reoxidized crystals of ferrocytochrome c confirm the absence of a conformation change. A detailed analysis of hydrogen bonding shows the presence of six beta or 310 bends of type II with obligatory glycines in the 3rd residue position. This explains 6 of the 10 nearly invariant glycines in the molecule. Close packing contacts account for three more, and only the invariant glycine 1 remains a mystery.
Amino Acid Sequence↗
Molecular packing of collagen: three-dimensional analysis of electrostatic interactions.
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Amino Acid Sequence↗
The crystal structure of 10-methylisoalloxazine hydrobromide dihydrate.
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Bromides↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗