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R Josephs

Publications and source records attributed to R Josephs.

At least 37 records · Page 2Linked to original sources
PubMed1991

The hand of the helix of deoxyhemoglobin S fibers.

Electron micrographs of deoxyhemoglobin S fiber cross sections provide an end-on view of the fiber whose appearance is sensitive to small changes in orientation. We have developed a procedure to exploit this sensitivity in order to determine the hand of these particles. In a sickle hemoglobin fiber the hemoglobin molecules form long pitch helical strands which twist about the particle axis with a pitch of about 3000 A. Tilting a 400-A-thick cross section by a few degrees aligns one of the long pitch helices so that it is nearly parallel to the direction of view. When a strand of hemoglobin molecules in a fiber is aligned in this manner it appears as a strongly contrasted bright spot. It is this spot, rather than the fiber axis, which appears to be the apparent center of rotation of the cross section. The direction of the displacement of the spot from the particle axis depends upon the particle hand and tilt direction. We have used this property to determine that sickle hemoglobin fibers are right-handed particles. This method may be applicable to other particles with long pitch helices as well.

Computer Simulation↗
PubMed1990

Length distributions of hemoglobin S fibers.

Electron microscopy of sickle cell hemoglobin fibers fixed at different times during gelation shows an exponential distribution of fiber lengths, with many short fibers and few long ones. The distribution does not change significantly with time as polymerization progresses. If this distribution of lengths reflects kinetic mechanism of fiber assembly, it complements information from studies of the progress of average properties of the polymers and, as has been done for other rod-like polymerizing systems, permits testing of models for the mechanism of fiber assembly. In this case, the results are consistent with the double nucleation model of Ferrone et al. or with a related alternative model based on fiber breakage. However, other possible causes of this microheterogeneity exist, including: breakage due to solution shearing of the long, rod-like, fibers; the presence of residual nuclei; equilibrium relations governing polymerization; and breakage of solid-like but weak gels that develop early and adhere to the grid. The arguments against the first three of these possibilities suggest that they are not responsible. However, breakage of entanglements or cross-links in a solid-like and adherent gel is consistent with the distributions.

Hemoglobin, Sickle↗
PubMed1990

On the structure of erythrocyte spectrin in partially expanded membrane skeletons.

Spectrin is generally believed to play an important role in the erythrocyte membrane's ability to deform elastically. We have studied the structure of negatively stained spectrin in partially expanded membrane skeletons to determine how its molecular structure confers elastic properties on the cell membrane. Fourier analysis of electron micrographs of spectrin reveals that the alpha and beta subunits are twisted about a common axis, forming a two-start helix with twofold rotational symmetry. We propose that elastic deformation of the cell is mediated by transient extension of the helix by mechanical forces.

Computer Simulation↗
PubMed1989

Intermolecular contacts within sickle hemoglobin fibers.

By combining X-ray crystallographic co-ordinates of sickle hemoglobin (HbS) molecules with three-dimensional reconstructions of electron micrographs of HbS fibers we have synthesized a model for the structure of the clinically relevant HbS fiber. This model largely accounts for the action of 55 point mutations of HbS whose effect on fiber formation has been studied. In addition, it predicts locations at which additional point mutations are likely to affect fiber formation. The number of intermolecular axial contacts decreases with radius until, at the periphery of the fiber, there are essentially no axial contacts. We suggest that this observation accounts for the limited radial growth of the HbS fiber and that a similar mechanism may be a factor in limiting the size of other helical particles. The methodology for the synthesis of the fiber model is applicable to other systems in which X-ray crystallographic and electron microscopic data are available.

Crystallography↗
PubMed1989

On the assembly of sickle hemoglobin fascicles.

Deoxyhemoglobin S fibers associate into bundles, or fascicles, that subsequently crystallize by a process of alignment and fusion. We have used electron microscopy to study the formation of fascicles and the changes in fiber packing that occur during the conversion of fascicles to crystals. The first event in crystallization involves fibers forming fascicles that are initially small and poorly ordered but, with time, become progressively larger and more highly ordered. After six to eight hours, the fibers in a fascicle form a crystalline lattice. The three-dimensional unit cell parameters of this lattice are a = 1300 A, b = 365 A, and c = 210 A (the a axis is parallel to the fiber axis). Fibers have an elliptical cross-section whose major and minor axes are 250 A and 185 A, respectively. When projected on to the unit cell vectors, these dimensions are 210 A and 155 A, so the unit cell dimension of 365 A implies that there are two fibers per unit cell. Theoretically, fibers could pair so that each member of the unit cell is oriented in the same direction (parallel) or opposite directions (antiparallel). Fourier transforms of electron micrographs (or models) cannot distinguish between these alternatives, since the two arrangements produce very similar intensity distributions. The orientation of the fibers was determined from cross-sections of the fascicles in which the fibers are seen end-on. In this view the images of the fibers are rotationally blurred because the fibers twist 30 degrees to 40 degrees about their helical axis through the 300 A to 400 A thick section. We have been able to remove the rotational blur from each of the fibers in the unit cell using the procedures described by Carragher et al. The deblurred images of the two fibers in the unit cell are related by mirror symmetry. This relationship means that the fibers are antiparallel. These observations suggest that crystallization of fibers in fascicles is mediated by assembly of the fibers into antiparallel pairs that contain equal numbers of double strands running in each direction.

Crystallization↗
PubMed1988

Structural analysis of polymers of sickle cell hemoglobin. I. Sickle hemoglobin fibers.

The structure of fibers of deoxyhemoglobin S has been under investigation for several years and a number of different models have been proposed for the arrangement of molecules within the particles. We have used reconstruction and modeling techniques in our analysis of these structures. Several new approaches have been employed in this analysis in order to provide improved estimates of the co-ordinates, pairing, and polarity of the hemoglobin S molecules. Fibers have a variable pitch and, in order to minimize distortions in the reconstructed density maps associated with these variations in pitch, we have developed an iterative procedure to measure the instantaneous pitch and have modified the reconstruction algorithm to incorporate the measured values. This procedure improves the accuracy with which the hemoglobin S molecules can be located in the density maps. Furthermore, the determination of the instantaneous pitch allows us to measure directly the rotation of the individual hemoglobin molecules. These measurements are in excellent agreement with the values predicted using a random angular walk model (as originally proposed for F-actin) to describe the variable pitch. The reconstructions confirm that the fiber consists of 14 strands of hemoglobin S arranged in a hexagonally shaped cross-section. We have determined the pairing of the molecules to form double strands directly from the density maps by identifying the molecules that have intermolecular distances that conform to those of double strands in the Wishner-Love crystal. The seven double strands identified in this manner are consistent with the strand pairings proposed by Dykes et al. (1979) rather than the alternate pairings proposed by Rosen & Magdoff-Fairchild (1985). In addition, we have for the first time determined the polarity of the double strands directly from the reconstruction data. This was achieved using a procedure that amounts to essentially "dissecting" individual double strands from the reconstructed density maps so that their density distribution could be examined independently of the neighboring double strands. Knowledge of the relative polarities of the double strands is essential for determining the intermolecular interactions that stabilize the fiber.

Crystallography↗
PubMed1988

Structural analysis of polymers of sickle cell hemoglobin. II. Sickle hemoglobin macrofibers.

Sickle cell hemoglobin macrofibers are an important intermediate in the low pH crystallization pathway of deoxygenated hemoglobin S that link the fiber to the crystal. Macrofibers are a class of helical particles differing primarily in their diameters but are related by a common packing of their constituent subunits. We have performed three-dimensional reconstructions of three types of macrofibers. These reconstructions show that macrofibers are composed of rows of Wishner-Love double strands in an arrangement similar to that in the crystal. We have measured the orientation and co-ordinates of double strands in macrofibers using cross-correlation techniques. In this approach, the electron density projections of double strands calculated from the known high-resolution crystal structure are compared with regions along the length of the particles in which the distinct pattern of double strands in c-axis projection may be observed. Contrary to assertions by Makinen & Sigountos (1984), our results unambigously demonstrate that adjacent rows of double strands in macrofibers are oriented in an antiparallel manner, as in the Wishner-Love crystal. Adjacent rows of antiparallel double strands are displaced along the helical axis relative to their co-ordinates in the crystal. Electron density models of macrofibers based on the crystallographic structure of the sickle hemoglobin double strand are in good agreement with the projections of macrofibers observed in electron micrographs. We have studied the structure of a closely related crystallization intermediate, the sickle hemoglobin paracrystal. The arrangement of double strands in paracrystals is similar to that in Wishner-Love crystals, except that they are displaced along the a-axis of the crystal. Measurements of the double strand co-ordinates reveal that the distribution of strand positions is bimodal. These results further establish the close structural relationship between macrofibers and paracrystals as intermediates in the crystallization of deoxygenated sickle hemoglobin.

Computer Simulation↗
PubMed1988

Structural analysis of polymers of sickle cell hemoglobin. III. Fibers within fascicles.

We have examined the structure of hemoglobin S fibers, which are associated into large bundles, or fascicles. Electron micrographs of embedded and cross-sectioned fascicles provide an end-on view of the component fibers. The cross-sectional images are rotationally blurred as a result of the twist of the fiber within the finite thickness of the section. We have applied restoration techniques to recover a deblurred image of the fiber. The first step in this procedure involved correlation averaging images of cross-sections of individual fibers in order to improve the signal-to-noise ratio. The rotationally blurred image was then geometrically transformed to polar co-ordinates. In this space, the rotational blur is transformed into a linear blur. The linearly blurred image is the convolution of the unblurred image and a point spread function that can be closely approximated by a square pulse. Deconvolution in Fourier space, followed by remapping to Cartesian co-ordinates, produced a deblurred image of the original micrograph. The deblurred images indicate that the fiber is comprised of 14 strands of hemoglobin S. This result provides confirmation of the fiber structure determined using helical reconstruction techniques and indicates that the association of fibers into ordered arrays does not alter their molecular structure.

Hemoglobin, Sickle↗
PubMed1988

The reconstruction of helical particles with variable pitch.

In the reconstruction of helical particles, it is normally assumed that translation along the length of a particle is coupled to rotation about its axis. This assumption is not valid for particles whose pitch varies along the particle length (e.g. actin, HbS fibers), and application of the usual algorithms results in significant errors in both the shape and coordinates of subunits in the reconstructed density map. We have developed an iterative procedure for reconstructing particles with variable pitch. The goal of this procedure is to obtain an accurate estimate of the local pitch of the particle which can then be incorporated into the reconstruction algorithm. This involves synthesis of trial model structures which have constant pitch. The local pitch is derived from a cross-correlation analysis between these trial models and the variable pitch particles. The constant pitch models are constructed using coordinates measured from the reconstructed density maps. Each iteration of the procedure provides an improved estimate of the pitch which is incorporated into the succeeding iteration. The fidelity of the reconstruction is determined from cross-correlation between the original micrograph and a variable pitch model. The iterations are continued until the cross-correlation coefficient between the variable pitch model and the micrograph of the particle is maximized. The implementation of the iterative procedure is described and its behavior is evaluated using model structures which incorporate variations in pitch similar to those actually occurring in sickle hemoglobin fibers. The results indicate that the iterative reconstruction procedure considerably reduces the errors associated with constant pitch reconstructions. These tests provide a basis for applying this procedure in the structural analysis of micrographs of helical particles which display variable pitch. Application to sickle hemoglobin fibers resulted in an improvement in the accuracy with which the hemoglobin S molecules can be located in the density maps.

Algorithms↗
PubMed1987

The three-dimensional structure of sickle hemoglobin macrofibers.

The crystallization of deoxygenated sickle cell hemoglobin (HbS) at pH less than 6.5 is preceded by the formation of HbS macrofibers. In stirred solutions, macrofibers appear several hours after the formation of fibers and subsequently aggregate and crystallize. Like fibers, macrofibers are long helical structures consisting of alternating narrow and wide regions. Macrofibers have a pitch of 10,000 A and a diameter averaging 500 A. Here we present a three-dimensional structural analysis of HbS macrofibers. Image reconstructions of these particles show that they consist of 5 antiparallel rows with 8, 11, 12, 11, and 8 double strands per row. Double strands in the outer two rows are asymmetrically disposed resulting in "notches" in opposite corners of the particle cross section. The notches produce characteristic features in electron micrographs of macrofibers. Fourier-filtered images of macrofibers have been produced which reveal their structure in considerable detail. Computed Fourier transforms of macrofibers exhibit spacings of 64 A, 185 A, and 53 A similar to those in the Wishner-Love crystal. A cross-correlation analysis between filtered macrofiber images and electron density projections of Wishner-Love double strands establishes that double strands in adjacent rows are antiparallel. This analysis also shows that the macrofiber rows are displaced axially by differing amounts depending on their distance from the helical axis. Using this data, we have computed a three-dimensional electron density model of the macrofiber based on atomic hemoglobin coordinates. This model reproduces in detail the many features revealed in Fourier-filtered macrofiber images.

Computer Simulation↗
PubMed1986

The restoration of electron micrographs blurred by drift and rotation.

We have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation. Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane. A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise. Model images were also used to investigate how an incorrect estimate of the point spread function describing the blur would effect the restoration. Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified. In the case of the rotationally blurred images this procedure was accomplished by transforming the image to polar coordinates. The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase. The quality of the restoration was judged by comparisons of the restored images to undegraded images. Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring.

Algorithms↗
PubMed1986

Ultrastructure of the intact skeleton of the human erythrocyte membrane.

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

Actin Cytoskeleton↗
PubMed1984

Macrofiber structure and the dynamics of sickle cell hemoglobin crystallization.

Fibers of deoxyhemoglobin S undergo spontaneous crystallization by a mechanism involving a variety of intermediate structures. These intermediate structures, in common with the fiber and crystal, consist of Wishner-Love double strands of hemoglobin S molecules arranged in different configurations. The structure of one of the key intermediates linking the fiber and crystal, called a macrofiber, has been studied by a variety of analytical procedures. The results of the analysis indicate that the intermediates involved in the fiber to crystal transition have many common structural features. Fourier analysis of electron micrographs of macrofibers confirms that they are composed of Wishner-Love double strands of hemoglobin molecules. Electron micrographs of macrofiber cross-sections reveal that the arrangement of the double strands in macrofibers resembles that seen in micrographs of the a axis projection of the crystal. This orientation provides an end-on view of the double strands which appear as paired dumb-bell-like masses. The structural detail becomes progressively less distinct towards the edge of the particle due to twisting of the double strands about the particle axis. Serial sections of macrofibers confirm that these particles do indeed rotate about their axes. The twist of the particle is right handed and its average pitch is 10,000 A. The effect of rotation on the appearance of macrofiber cross-sections 300 to 400 A thick can be simulated by a 15 degrees rotation of an a axis crystal projection. The relative polarity of the double strands in macrofibers and crystals can be determined easily by direct inspection of the micrographs. In both macrofibers and crystals they are in an anti-parallel array. On the basis of these observations we conclude that crystallization of macrofibers involves untwisting and alignment of the double strands.

Crystallization↗
PubMed1984

Ultrastructure of unit fragments of the skeleton of the human erythrocyte membrane.

We have examined fragments of the filamentous network underlying the human erythrocyte membrane by high-resolution electron microscopy. Networks were released from ghosts by extraction with Triton X-100, freed of extraneous proteins in 1.5 M NaCl, and collected by centrifugation onto a sucrose cushion. These preparations contained primarily protein bands 1 + 2 (spectrin), band 4.1 and band 5 (actin). The networks were partially disassembled by incubation at 37 degrees C in 2 mM NaPi (pH 7), which caused the preferential dissociation of spectrin tetramers to dimers. The fragments so generated were fractionated by gel filtration chromatography and visualized by negative staining with uranyl acetate on fenestrated carbon films. Unit complexes, which sedimented at approximately 40S, contained linear filaments approximately 7-8 nm diam from which several slender and convoluted filaments projected. The linear filaments had a mean length of 52 +/- 17 nm and a serrated profile reminiscent of F-actin. They could be decorated in an arrowhead pattern with S1 fragments of muscle heavy meromyosin which, incidentally, displaced the convoluted filaments. Furthermore, the linear filaments nucleated the polymerization of rabbit muscle G-actin, predominantly but not exclusively from the fast-growing ends. On this basis, we have identified the linear filaments as F-actin; we infer that the convoluted filaments are spectrin. Spectrin molecules were usually attached to actin filaments in clusters that showed a preference for the ends of the F-actin. We also observed free globules up to 15 nm diam, usually associated with three spectrin molecules, which also nucleated actin polymerization; these may be simple junctional complexes of spectrin, actin, and band 4.1. In larger ensembles, spectrin tetramers linked actin filaments and/or globules into irregular arrays. Intact networks were an elaboration of the basic pattern manifested by the fragments. Thus, we have provided ultrastructural evidence that the submembrane skeleton is organized, as widely inferred from less direct information, into short actin filaments linked by multiple tetramers of spectrin clustered at sites of association with band 4.1.

Cell Fractionation↗