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J M Carazo

Publications and source records attributed to J M Carazo.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1984

General morphology and capsid fine structure of African swine fever virus particles.

The structure of African swine fever virus particles has been examined by electron microscopy. The analysis of virions prepared by negative staining, thin sectioning, and freeze-drying and shadowing showed that the virus particle was composed of several concentric structures with an overall icosahedral shape. The inner region of the virus particles was a nucleoid that was surrounded by a membrane covered by the capsid. The capsid had side-to-side dimensions of 172 to 191 nm and was built up by capsomers arranged in an hexagonal lattice. Computer-filtered electron micrographs of either negatively stained or freeze-dried and shadowed capsids revealed capsomers with a hexagonal outline and a hole in the center. The intercapsomer distance ranged from 7.4 to 8.1 nm. The triangulation number of the capsid was estimated to be 189 to 217, indicative of 1892 to 2172 capsomers. Extracellular African swine fever virus particles had an external membrane that resembled the cytoplasmic unit membrane.

African Swine Fever Virus↗
PubMed1984

Structural study of tetragonal-ordered aggregates of phage phi 29 necks.

A new class of two-dimensional tetragonal aggregates of phage phi 29 necks has been studied by electron microscopy and a combination of Fourier filtering procedures and detailed rotational analysis. The results confirm the main features of the head-to-tail connecting region previously observed in hexagonal aggregates. There are several differences in the resulting pictures that can be attributed to the different way in which the aggregates are organized and stained.

Bacteriophages↗
PubMed1983

Structural localization of the proteins of the head to tail connecting region of bacteriophage phi 29.

The head to tail connector of bacteriophage phi 29 has been studied to locate its two structural proteins (p10 and p11). Treatment with trypsin led to proteolysis of p10 while p11 remained intact. Computer filtration of electron micrographs of crystals of trypsinized necks showed a change in the external 12-fold area of the neck when compared with control necks. Proteolized necks completely released p10 after treatment with low concentrations of an ionic detergent. The resulting structures, containing p11, showed similarity with the central area of the neck (seen in front view) and accounted for the lower collar and the axial extension of the neck (seen in side view). These results, together with the differences found in the proteolysis of p10 in necks with and without appendages, lead to a model for the neck region of phi 29 in which p10 makes the upper collar (and the external 12-fold area of the neck seen in front view), while p11 forms the lower collar and the axial extension (the inner region of the neck seen in front view).

Bacillus subtilis↗
PubMedDate not supplied

A structural model for the Escherichia coli DnaB helicase based on electron microscopy data.

The DnaB protein is the major replicative DNA helicase in Escherichia coli. It hydrolyzes ATP to promote its translocation in the 5' to 3' direction on single-stranded DNA templates, facilitating the separation of strands of duplex DNA in its path. This places it on the lagging strands at replication forks during chromosomal DNA replication. Electron microscopic images of negatively stained DnaB protein have been studied and processed to produce a three-dimensional reconstruction of the protein oligomer at 2.7 nm resolution. While it is known that the native protein is a complex of six identical 52-kDa subunits, the specimen shows threefold rather than sixfold symmetry, with three outer stain-excluding regions surrounding another six, more massive, lobules. There is a channel through the particle that appears fully open on both sides. Based on these results, a structural model for the oligomer is presented, and functional implications are considered.

Bacterial Proteins↗
PubMedDate not supplied

Proposal for a new distributed database of macromolecular and subcellular structures from different areas of microscopy.

In this work we address the problem of information access that arises in the field of three-dimensional structure determination, by means of image processing, from data obtained by various types of microscopy. A prototype of a distributed database containing three-dimensional structural information is presented. In this database the volume information is linked, if possible, to other sources of catalogued information such as sequence data, atomic coordinates, and bibliographies. The solution we propose is sufficiently general to be applicable to data in other fields of biomedical science.

Computer Communication Networks↗