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B L Trus

Publications and source records attributed to B L Trus.

At least 55 records · Page 3Linked to original sources
PubMed1991

Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays.

The mitochondrial outer membrane contains a protein, called VDAC, that forms large aqueous pores. In Neurospora crassa outer membranes, VDAC forms two-dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (C. Mannella, Science 224, 165, 1984). Fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each other. Most likely, this technique has revealed both surfaces of the same two-dimensional crystal with lattice parameters: a = 12.3 +/- 0.1 nm, b = 11.2 +/- 0.1 nm, and theta = 109 +/- 1 degree. Three-dimensional reconstructions of the surface reliefs on both sides of the crystal show them to be very similar. The majority of the protein forming the channel appears to be at or below the level of the membrane. To address the issue of the number of 30-kDa polypeptides that form a VDAC channel, measurements of mass per unit area were carried out by analyzing scanning transmission electron micrographs of unstained, freeze-dried arrays. The crystal form used for mass analysis contained the same motif of six stain-accumulating centers per unit cell, with p2 symmetry as in the oblique configuration, but it had a different orientation relative to the lattice lines. These data yielded a surface density of 1.9 +/- 0.2 kDa/nm2, indicating that there is a one-to-one ratio between VDAC polypeptides and the channels visualized in filtered electron micrographs, and that VDAC membrane crystals contain 68% protein and 32% lipid by mass.

Crystallization↗
PubMed1991

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.

Gene Products, env↗
PubMed1990

Molecular structure of the cell-attachment protein of reovirus: correlation of computer-processed electron micrographs with sequence-based predictions.

The receptor-recognition interaction that initiates reovirus infection is mediated by the sigma 1 protein, located at the vertices of the icosahedral virion. We have applied computer-based image-averaging techniques to electron micrographs of negatively stained preparations of sigma 1 purified from virions (serotype 2 Jones). Combining these results with inferences based on the amino acid sequence has led to a molecular model in which the overall folding of the chains is described; its conformation embodies motifs, coiled-coil alpha-helices and nodular multichain elements rich in beta-sheets, previously detected in the corresponding proteins of other viruses, but with some novel variations. Sigma 1 is a filamentous lollipop-shaped molecule with an overall length of approximately 48 nm; it has a flexible "tail," approximately 40 nm long by 4 to 6 nm wide, terminating at its distal end in a globular "head," approximately 9.5 nm in diameter. The purified protein is a tetramer (4 by 50 kilodaltons) consisting of two similarly oriented dimers bonded side by side and in register. For each chain, a cluster of hydrophobic residues at its amino terminus resides at the proximal end of the tail; next, an alpha-helical domain (residues 25 to 172) participates in a two-chained coiled coil, 22 nm long, with two such coiled coils pairing laterally to form the proximal half of the tail. The remainder of the tail (residues 173 to approximately 316) is less uniform in width and is expected to be rich in beta-sheet; the interdimer bonding is evidently sustained through this portion of the molecule. Finally, the globular head consists of the carboxy-terminal domains (which contain the receptor-binding sites) folded into compact globular conformations; in appropriate side views, the head is resolved into two subunits, presumably contributed by the respective dimers. This model for how the four sigma 1 polypeptide chains are threaded in parallel through the fiber is supported by the observed match between an empirical curvature profile, which identifies the locations of relatively flexible sites along the tail, and the flexibility profile predicted on the basis of the model. Appraisal of the interactions that stabilize the coiled coils suggests that (i) the alpha-helices are individually only marginally stable, a property that may be of significance with regard to the retracted conformation in which sigma 1 is accommodated in the intact virion, and (ii) the predominant interactions between the two coiled coils are likely to involve hydrogen bonding between patches of uncharged residues.

Amino Acid Sequence↗
PubMed1990

Computerized cataract detection and classification.

156 eyes of patients and normal volunteers were classified at the slit lamp into the following pure groups: normal (n = 50), nuclear (n = 39), cortical (n = 33) and PSC (n = 34). The eyes were photographed with the Topcon SL-45 Scheimpflug camera and the images scanned and processed to obtain one dimensional profiles through a 40 x 440 micron axial window. Of the 156 profiles, 90 were used as reference samples and were processed to obtain average profiles. The remaining 66 "unknown" profiles and each of the reference profiles, were classified into the four groups based on their distance from the average profiles in Euclidean space. The system was found to be very sensitive (98%) in detecting the presence of cataracts and specific (100%) in identifying normal, i.e. cataract negative lenses. In classifying pure cataracts into the various classes 98% of answers were correct.

Adult↗
PubMed1989

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

Actins↗
PubMed1989

An introduction to image processing in medical microscopy.

Image processing hardware and software have evolved in the past few years into powerful, relatively inexpensive tools for micrographic research. Although now within the reach of most researchers, image processing techniques appear under-utilized based on a review of current biomedical literature. This article will give a basic overview of image processing as applied to both light and electron micrographs, citing current applications.

Animals↗
PubMed1988

Naturally crystalline porin in the outer membrane of Bordetella pertussis.

The Gram-negative bacterium Bordetella pertussis is the agent responsible for whooping-cough, and much interest has focused on the functions, structures and immunological properties of the molecules exposed at its outer surface. We have found by electron microscopy that cells of two strains of B. pertussis are covered with a crystalline surface lattice. This lattice is not an extrinsic layer of high molecular weight glycoproteins, such as occur on many other bacteria, but is a natural crystal of an intrinsic membrane protein of 40,000 Mr. This molecule has been shown to be an anion-selective member of an extensive family of proteins ("porins") that render Gram-negative outer membranes permeable to solutes of up to approximately 650 Mr. Computer image processing reveals a trimeric channel-like structure that closely resembles other porins visualized in artificial arrays after treatment with detergents, but in a novel (p2) crystal form. This correlation provides a "missing link" between earlier structural studies based on artificial arrays of porins (of undefined physiological status), and membrane-permeabilization experiments with solubilized porins (in undefined structural states). For the strains characterized so far, crystallinity of the porin surface lattice shows an intriguing correlation with nonpathogenicity.

Bacterial Outer Membrane Proteins↗
PubMed1988

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes.

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.

Amino Acid Sequence↗
PubMed1988

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7.

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Amino Acid Sequence↗
PubMed1988

Membrane contents of distinct subpopulations of coated vesicles determined by scanning transmission electron microscopy.

In order to investigate the heterogeneity of clathrin-coated vesicles purified from rat liver, and to quantitate rigorously their membrane contents, we have analyzed scanning transmission electron micrographs of unstained coated vesicles before and after extraction with the non-ionic detergent Triton X-100, as well as of vesicles whose coats had been removed by dialysis against 10 mM or 100 mM Tris (pH 8.2). Their respective distributions of particle masses were thus determined and compared, in light of complementary biochemical quantitations of lipid and protein. Smaller coated particles, 25-45 MDa in mass and 60-80 nm in diameter, lose no mass when extracted with Triton, and disappear when their coats are dissociated. We conclude that they do not contain membrane vesicles, although they have dense, presumably proteinaceous, cores. They may represent particles generated during tissue homogenization or, possibly, a storage form of clathrin. The remaining 70% contain bona fide vesicles: these particles are 75-150 nm in diameter, and their average mass is about 80 MDa, of which 48 MDa is contributed by coat proteins, 10-12 MDa by phospholipid and cholesterol, and 20-22 MDa by vesicle-associated proteins. Their vesicles are of two types: smaller, denser, vesicles that contain substantial amounts of internalized material, and larger, less dense, vesicles that do not. The distinction between them may, in view of other findings, reflect a difference between coated vesicles derived respectively from the Golgi and the plasma membrane.

Animals↗
PubMed1988

Conformational characteristics of the complete sequence of group A streptococcal M6 protein.

M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were "core" nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.

Amino Acid Sequence↗
PubMed1987

A new resolution criterion based on spectral signal-to-noise ratios.

A new criterion for the "useful" resolution of electron micrographs of macromolecular particles is introduced. This criterion is based on estimation of the spatial frequency limit beyond which the spectral signal-to-noise ratio (SSNR) falls below an acceptable baseline. Applicable to both periodic and aperiodic specimens, this approach is particularly apposite for sets of correlation-averaged images. It represents a straightforward and intuitively appealing generalization of the traditional method of estimating the resolution of crystalline specimens from the spectral ranges of periodic reflections in their diffraction patterns. This method allows one to assess how closely the resolution of an averaged image based on N individual images approaches the ultimate resolution obtainable from an indefinitely large number of statistically equivalent images. Inter-relationships between the SSNR and two other measures of resolution, the differential phase residual and the Fourier ring correlation coefficient, are discussed, and their properties compared.

Biopolymers↗
PubMed1987

In vivo studies on cataracts using the Scheimpflug slit lamp camera.

We conducted reproducibility studies on an in vivo objective method of documenting cataracts: Scheimpflug photography using the Topcon SL 45 camera. Normal and cataractous lenses were photographed and the photographs digitized and analyzed using a Perkin Elmer microdensitometer attached to a PDP 11 and Vax 11/780 computer. Linear densitometry was performed through the center of the lens. We found very good reproducibility. The intraclass correlation for the mean densities in the nucleus was 0.95: that is, intra- and interobserver variability accounted for only 5% of the overall variability. For the anterior cortex, intraclass correlation was 0.88 and for posterior cortex it was 0.84. This method may be useful, within limits, in future clinical trials of anticataract medications.

Cataract↗
PubMed1986

Molecular mobility in the gap regions of type I collagen fibrils.

Recent studies of the structure of Type I collagen fibrils (Piez and Trus, Biosci. Rep. 1:801-810, 1981; Fraser, MacRae, Miller and Suzuki, J. Mol. Biol. 167:497-521, 1983) suggest that the segments of the collagen molecule which comprise the gap region are more mobile than those which comprise the overlap region. We have analyzed the distribution of amino acid residues and triplet types between the two regions, and find significantly non-uniform distributions for Ala, Gln, His, Hyp, Leu, Phe, and Tyr, and for triplets containing two imino acid residues. Taken together with the lower packing density in the gap region these observations provide a basis for understanding the greater mobility of the molecular segments in the gap region. In addition, we have examined the linear distribution of residue types in the two regions and also the hydropathy profile (Kyte and Doolittle, J. Mol. Biol. 157: 105-113, 1982). These reveal a segment of the gap region comprising helical residues 165-173, 399-407, 633-641 and 867-975 which has the highest hydropathy index, is devoid of charged residues, and contains very high proportions of Ala, Hyp and Phe.

Amino Acid Sequence↗
PubMed1986

Odd men out: a quantitative objective procedure for identifying anomalous members of a set of noisy images of ostensibly identical specimens.

Achievement of an optimal improvement in signal-to-noise ratio from image averaging techniques depends crucially on the assumption that all members of the set of images to be averaged are fundamentally alike. In HREM of biological macromolecules, this assumption may be invalid for such reasons as variations in viewing geometry, non-uniformity of staining, or structural perturbations caused by specimen preparation procedures of radiation damage. Inclusion of data that are compromized these or other factors will degrade the information content of the averaged image. Here we present an algorithm which provides an objective quantitative method for the identification and elimination of anomalous members of a set of pre-aligned images. Based on a statistical criterion of mutual consistency, the algorithm forms an ordered list in which the individual images are ranked from most to least reliable. On specification of the noise statistics--in the formulation given here, of stationary white noise--an acceptability threshold in this ordered list is imposed. The derivation and implementation of this algorithm are presented, its properties discussed, and its application illustrated using both real and model electron micrograph data.

Algorithms↗
PubMed1986

Helical structure of Bordetella pertussis fimbriae.

The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae. The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix. This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens. These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6). Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy. The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals. Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical. From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria. It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae.

Bordetella pertussis↗
PubMed1986

Hexavalent capsomers of herpes simplex virus type 2: symmetry, shape, dimensions, and oligomeric status.

The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface.

Capsid↗