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J M Carazo

Publications and source records attributed to J M Carazo.

At least 19 recordsLinked to original sources
PubMed1992

Three-dimensional structure of T3 connector purified from overexpressing bacteria.

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.

Capsid↗
PubMed1992

Detection, classification and 3D reconstruction of biological macromolecules on hypercube computers.

In this work we present results of the mapping on hypercube computers of some of the key steps involved in the procedure for 3D structural determination from transmission electron microscopy images. The goal is the introduction of parallel processing tools in the field of electron microscopy image processing. We show how the rich topology of the hypercube, combined with an efficient programming strategy, allows for order-of-magnitude increase in computational capacity for such time-consuming tasks as calculation of multidimensional FFT's, cross-correlation coefficients, fuzzy partitioning functionals and the filtered back-projection 3D reconstruction method.

Algorithms↗
PubMed1991

Electron microscopy study of GroEL chaperonin: different views of the aggregate appear as a function of cell growth temperature.

We have studied two members of the family of morphogenetic factors or chaperonins, the GroEL-like factors from Escherichia coli and Bacillus subtilis, in order to determine the possible structural basis of their related function in promoting the correct and efficient assembly of biological oligomers. The main objective of this work has been to study by transmission electron microscopy the possible changes that these factors may undergo when subjected to a number of different conditions such as changes in temperature in vivo and in pH in vitro. We applied both rotational and multivariate statistical analyses of single particles to images of GroEl-like aggregates from the two bacteria. The most striking result is the finding of two distinct "front views" of these aggregates, from both E. coli and B. subtilis. One view, which has not been described earlier, shows a sixfold symmetry and is most abundant at growing temperatures below 37 degrees C. After heat shock, a view showing seven morphological units becomes dominant. On the basis of our analysis it is clear that GroEL-like morphogenetic factors from two unrelated bacteria such as E. coli and B. subtilis present two distinct views: one sixfold and the other sevenfold. Their relative percentage of appearance is related to the temperature at which the cells were grown and also to the storage conditions (pH).

Bacillus subtilis↗
PubMed1990

Fuzzy sets-based classification of electron microscopy images of biological macromolecules with an application to ribosomal particles.

Pattern recognition methods based on the theory of fuzzy sets are tested for their ability to classify electron microscopy images of biological specimens. The concept of fuzzy sets was chosen for its ability to represent classes of objects that are vaguely described from the measured data. A number of partitional clustering algorithms and an extensive set of cluster-validity functionals (some already reported and some newly developed) have been applied to a test-data set and to two real-data sets of images. One of the real-data sets corresponded to images of the Escherichia coli 50S ribosomal subunits depleted of proteins L7/L12 and the other set to images of the E. coli 70S monosome in the range of overlap views. These two latter sets had been previously studied by another clustering methodology. The new results obtained by the application of fuzzy clustering techniques will be compared to those previously obtained and some conclusions about the consistency of these classifications will be drawn from this comparison.

Algorithms↗
PubMed1989

Three-dimensional reconstruction of the ribosome from Escherichia coli.

Three-dimensional image reconstruction has been applied to electron micrographs of noncrystalline, negatively stained ribosomes obtained from Escherichia coli. Several independent reconstructions all show an overall appearance resembling models that had been derived earlier by direct visual interpretation of electron micrographs. The reconstructed ribosomes show numerous structural details not recognized previously, some of which may be functionally significant. A large elongate cavity (approximately 8-nm long x 5-nm wide x 6-nm [maximal] deep) is present on the surface of the ribosome near the base of its stalk and is identifiable as a portion of a feature termed the interface canyon, which was detected in prior reconstructions of the large ribosomal subunit (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1107-1114). On the back of the ribosome, near the base of the central protuberance, is a hole leading to the interface canyon, which likely represents an exit site for the elongating polypeptide produced during protein biosynthesis. The exposed portion of the interface canyon appears well suited to bind two tRNA molecules in a configuration that is consistent with biochemical and structural data on the mechanism of peptide bond biosynthesis.

Escherichia coli↗
PubMed1989

Variations of the three-dimensional structure of the Escherichia coli ribosome in the range of overlap views. An application of the methods of multicone and local single-cone three-dimensional reconstruction.

Electron microscopic techniques are among the most important tools for obtaining structural information of biological specimens. However, the three-dimensional (3D) structural analysis of asymmetrical specimens that do not form crystalline sheets has traditionally presented serious methodological obstacles to its accomplishment. One of the fundamental questions to be addressed in this type of structural study is in what way, and to what degree, does the 3D structural conformation depend on the orientation of the specimen with respect to the electron microscopic support films. As a step in studying this problem, we have analyzed the variations of the 3D structure of the Escherichia coli 70S monosome by performing four different 3D reconstructions of the 70S monosome from subsets of images in the so-called overlap range of views. These subsets were selected according to a multivariate statistical analysis performed on the total population of overlap-range specimen images. A certain amount of structural variability exists among the 3D reconstructions, although many of the main morphological characteristics, as the relative orientation between the ribosomal subunits, remain unchanged. We have also generalized the random conical reconstruction technique (Radermacher, M., T. Wagenknecht, A. Verschoor, and J. Frank. 1987. J. Microsc. 146: 113-136) to include those cases where the specimen exhibits a rocking behavior with respect to the support. The resulting Multicone Reconstruction Technique has been applied to computer-generated images as well as the E. coli 70S monosome images from part of the overlap range of views.

Analysis of Variance↗
PubMed1988

Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12.

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

Escherichia coli↗
PubMed1988

Bacteriophage T3 connector: three-dimensional structure and comparison with other viral head-tail connecting regions.

The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution. The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter. There is a channel clearly defined in the narrower domain that almost closes along the wider domain. Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences. A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

Computer Graphics↗
PubMed1988

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging.

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.

Capsid↗
PubMed1988

Three-dimensional matching of macromolecular structures obtained from electron microscopy: an application to the 70S and 50S E. coli ribosomal particles.

In this work we present a general computational method capable of finding the relative orientation of two structures represented by samples on a three-dimensional grid. It is shown that the three-dimensional shift and the three independent rotations necessary for the correct relative spatial placement of the two volumes can be obtained either from the auto-correlation function of the volumes or from a direct cross-correlation, depending on the specific problem to be solved. This method has been applied to the problem of fitting the 50S ribosomal subunit into the 70S monosome from E. coli, structures that were available as three-dimensional reconstructions from electron microscopical data.

Escherichia coli↗
PubMed1988

Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli.

Images of macromolecules obtained in the electron microscope are subjected to correspondence analysis. The structure inherent in the data in the resulting low-dimensional factor space is characterized by a mixed classification method which combines the dynamic clouds clustering technique with hierarchical ascendant classification (HAC). For our data, the rejection of marginal clusters obtained by dynamic clouds clustering appears as a crucial prerequisite for a stable performance of HAC. The method is applied to two sets of 204 and 177 images that show the 70S ribosome of Escherichia coli, in the range of overlap views as defined by A. Verschoor and co-workers, and to two sets of 480 and 496 images of the 50S subunit of E. coli depleted of L7/L12 proteins in the well-defined crown view. Reproducible classes are obtained, which are characterized by images reconstituted from factorial coordinates. These classes appear to be related to different orientations on the specimen grid (in the case of the 70S particle) and to different conformational states (50S subunit).

Escherichia coli↗
PubMed1987

Restoration of direct Fourier three-dimensional reconstructions of crystalline specimens by the method of convex projections.

We consider the problem of the three-dimensional (3-D) reconstruction of objects by the direct Fourier method (DFM) and their restoration by the method of projections on to convex sets (POCS). The main discussion is centered on the case of specimens arranged in a two-dimensional (2-D) crystal and imaged by transmission electron microscopy, although the conclusions could be extended to more general cases. We present results of the restoration of the 3-D reconstruction of a computer generated 2-D crystal under different conditions of data collection limitation. A preliminary application with a real biological specimen (the connector of bacteriophage phi 29) is also presented. These results indicate that POCS can be used practically, in certain cases, to restore 3-D reconstructions obtained by the DFM, giving grounds for the proposal of the study of a combined DFM + POCS (reconstruction + restoration) method for the determination of biological structures by electron microscopy and 3-D image processing.

Coliphages↗
PubMed1986

Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution.

The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.

Bacteriophages↗
PubMed1986

Computer graphic display method for visualizing three-dimensional biological structures.

A computer graphic display method that produces two-dimensional perspective views of three-dimensional objects is presented. The method is applied to the reconstruction at a resolution of 2.2 nanometers of the neck of bacteriophage phi 29, obtained from transmission electron micrographs processed by the direct Fourier method. The combined use of directed illumination, reflectance models, color, and different levels of transparency provides a powerful tool for a better interpretation of the three-dimensional structure, allowing improved correlation with genetic, structural, and biochemical data.

Bacteriophages↗
PubMed1986

Bacteriophage T3 gene 8 product oligomer structure.

The structure of the connector of bacteriophage T3 (built up by the product of gene 8) has been studied in two dimensions by combined use of translational and rotational image filtering procedures applied to tetragonal ordered aggregates of the former oligomers. This analysis, performed up to 1/1.6 nm-1 resolution, has revealed the existence of a 12-fold symmetry in the outermost region of the specimen (mainly between radii 5.2 and 6.7 nm), a 6-fold one in the inner region (between radii 1.7 and 3.2 nm), and a hole in its center. These features are very similar to the ones described for the connectors of other phages, such as T4, lambda, and phi 29, thus suggesting a common mechanism for the functions carried out by this viral region.

Fourier Analysis↗
PubMed1985

Three-dimensional reconstruction of bacteriophage phi 29 neck particles at 2 X 2 nm resolution.

The three-dimensional structure of the head-to-tail connecting region of bacteriophage phi 29 has been studied by analysing two-dimensional, hexagonal ordered aggregates of negatively stained viral necks to a resolution of 2 X 2 nm. These necks are composed of two proteins, p10 and p11; p10 being the connector protein. A 12-folded and a 6-folded axially symmetric domain are present in the specimen. The 12-folded domain is the larger part of the structure; it consists of 12 subunits associated in pairs. These subunits appear to be more closely paired towards the centre, where only six subunits are resolved forming the 6-folded domain. The pairs of subunits present an important twist between the 12-folded and the 6-folded areas. A conical hole is formed at the centre of the structure. This hole is more open at the 12-folded domain than at the level of the possible zone of interaction between p10 and p11, where it is almost closed. Protein p11 is very poorly represented in the reconstruction, probably due to lack of staining. The structure described for the phi 29 neck has many of the attributes expected for an active device involved in bacteriophage DNA encapsidation.

Bacteriophages↗
PubMed1985

Structure of phage phi 29 connector protein assembled in vivo.

The protein p10 that forms the connector of phage phi 29, has been produced in Escherichia coli harboring a plasmid that carried the gene coding for this protein. The connector protein is assembled in a 13.4-S oligomer that has an apparent molecular weight of 460,000, suggesting that it is a dodecamer. The purified oligomers have been studied by electron microscopy of the isolated particles as well as by image-processing techniques (Fourier and rotational filtering) of artificially induced two-dimensional aggregates. The results show that the purified p10 is assembled in a circular structure with a hole in its center and 12 morphological units in the periphery. Both the morphology and the dimensions of this p10 oligomer are very similar to those of the upper neck collar extracted from phi 29 viral particles. The results strongly suggest the close relationship between the p10 oligomers assembled in E. coli and the ones produced in phi 29 infected Bacillis subtilis.

Bacillus subtilis↗