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Biomedical subjects

J M Carazo

Publications and source records attributed to J M Carazo.

At least 19 recordsLinked to original sources
PubMed2006

Nonsmooth nonnegative matrix factorization (nsNMF).

We propose a novel nonnegative matrix factorization model that aims at finding localized, part-based, representations of nonnegative multivariate data items. Unlike the classical nonnegative matrix factorization (NMF) technique, this new model, denoted "nonsmooth nonnegative matrix factorization" (nsNMF), corresponds to the optimization of an unambiguous cost function designed to explicitly represent sparseness, in the form of nonsmoothness, which is controlled by a single parameter. In general, this method produces a set of basis and encoding vectors that are not only capable of representing the original data, but they also extract highly localized patterns, which generally lend themselves to improved interpretability. The properties of this new method are illustrated with several data sets. Comparisons to previously published methods show that the new nsNMF method has some advantages in keeping faithfulness to the data in the achieving a high degree of sparseness for both the estimated basis and the encoding vectors and in better interpretability of the factors.

Algorithms↗
PubMed2004

Designing and executing scientific workflows with a programmable integrator.

MOTIVATION: As in many other fields of science, computational methods in molecular biology need to intersperse information access and algorithm execution in a computational workflow. Users often find difficulties when transferring data between data sources and applications. In most cases there is no standard solution for workflow design and execution and tailored scripting mechanisms are implemented in a case by case basis. RESULTS: In this paper, we present a general purpose 'programmable integrator' that can access information from a variety of sources in a coordinated manner. Its usefulness in complex bioinformatics applications is claimed and supported by some application examples. AVAILABILITY: Tools are freely available to non-profit educations and research institutions. Usage by commercial organizations requires a license agreement. Software requirements: Java v1.3 (http://java.sun.com), Xerces XML Parser (http://xml.apache.org/xerces-j) and Kweelt implementation of XQuery (http://kweelt.sourceforge.net/).

Algorithms↗
PubMed2004

Transfer function restoration in 3D electron microscopy via iterative data refinement.

Three-dimensional electron microscopy (3D-EM) is a powerful tool for visualizing complex biological systems. As with any other imaging device, the electron microscope introduces a transfer function (called in this field the contrast transfer function, CTF) into the image acquisition process that modulates the various frequencies of the signal. Thus, the 3D reconstructions performed with these CTF-affected projections are also affected by an implicit 3D transfer function. For high-resolution electron microscopy, the effect of the CTF is quite dramatic and limits severely the achievable resolution. In this work we make use of the iterative data refinement (IDR) technique to ameliorate the effect of the CTF. It is demonstrated that the approach can be successfully applied to noisy data.

Algorithms↗
PubMed2004

A multiresolution approach to orientation assignment in 3D electron microscopy of single particles.

Three-dimensional (3D) electron microscopy (3DEM) aims at the determination of the spatial distribution of the Coulomb potential of macromolecular complexes. The 3D reconstruction of a macromolecule using single-particle techniques involves thousands of 2D projections. One of the key parameters required to perform such a 3D reconstruction is the orientation of each projection image as well as its in-plane orientation. This information is unknown experimentally and must be determined using image-processing techniques. We propose the use of wavelets to match the experimental projections with those obtained from a reference 3D model. The wavelet decomposition of the projection images provides a framework for a multiscale matching algorithm in which speed and robustness to noise are gained. Furthermore, this multiresolution approach is combined with a novel orientation selection strategy. Results obtained from computer simulations as well as experimental data encourage the use of this approach.

Algorithms↗
PubMed2004

XMIPP: a new generation of an open-source image processing package for electron microscopy.

X-windows based microscopy image processing package (Xmipp) is a specialized suit of image processing programs, primarily aimed at obtaining the 3D reconstruction of biological specimens from large sets of projection images acquired by transmission electron microscopy. This public-domain software package was introduced to the electron microscopy field eight years ago, and since then it has changed drastically. New methodologies for the analysis of single-particle projection images have been added to classification, contrast transfer function correction, angular assignment, 3D reconstruction, reconstruction of crystals, etc. In addition, the package has been extended with functionalities for 2D crystal and electron tomography data. Furthermore, its current implementation in C++, with a highly modular design of well-documented data structures and functions, offers a convenient environment for the development of novel algorithms. In this paper, we present a general overview of a new generation of Xmipp that has been re-engineered to maximize flexibility and modularity, potentially facilitating its integration in future standardization efforts in the field. Moreover, by focusing on those developments that distinguish Xmipp from other packages available, we illustrate its added value to the electron microscopy community.

Algorithms↗
PubMed2004

Normalizing projection images: a study of image normalizing procedures for single particle three-dimensional electron microscopy.

In the process of three-dimensional reconstruction of single particle biological macromolecules several hundreds, or thousands, of projection images are taken from tens or hundreds of independently digitized micrographs. These different micrographs show differences in the background grey level and particle contrast and, therefore, have to be normalized by scaling their pixel values before entering the reconstruction process. In this work several normalization procedures are studied using a statistical comparison framework. We finally show that the use of the different normalization methods affects the reconstruction quality, providing guidance on the choice of normalization procedures.

Algorithms↗
PubMed2003

A method for estimating the CTF in electron microscopy based on ARMA models and parameter adjustment.

In this work, a powerful parametric spectral estimation technique, 2D-auto regressive moving average modeling (ARMA), has been applied to contrast transfer function (CTF) detection in electron microscopy. Parametric techniques such as auto regressive (AR) and ARMA models allow a more exact determination of the CTF than traditional methods based only on the Fourier transform of the complete image or parts of it and performing some average (periodogram averaging). Previous works revealed that AR models can be used to improve CTF estimation and the detection of its zeros. ARMA models reduce the model order and the computing time, and more interestingly, achieve increased accuracy. ARMA models are generated from electron microscopy (EM) images, and then a stepwise search algorithm is used to fit all the parameters of a theoretical CTF model in the ARMA model previously calculated. Furthermore, this adjustment is truly two-dimensional, allowing astigmatic images to be properly treated. Finally, an individual CTF can be assigned to every point of the micrograph, by means of an interpolation at the functional level, provided that a CTF has been estimated in each one of a set of local areas. The user need only know a few a priori parameters of the experimental conditions of his micrographs, for turning this technique into an automatic and very powerful tool for CTF determination, prior to CTF correction in 3D-EM. The programs developed for the above tasks have been integrated into the X-Windows-based Microscopy Image Processing Package (Xmipp) software package, and are fully accessible at www.biocomp.cnb.uam.es.

Algorithms↗
PubMed2001

The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel.

Replicative helicases are motor proteins that unwind DNA at replication forks. Escherichia coli DnaB is the best characterized member of this family of enzymes. We present the 26 A resolution three-dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo-electron microscopy. Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC. The complex is arranged on the basis of interactions among DnaC and DnaB dimers. DnaC monomers are observed for the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C(3) architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through. We propose that the DnaB N-terminal domain is located at this face.

Bacterial Proteins↗
PubMed2000

pH-controlled quaternary states of hexameric DnaB helicase.

DnaB is the major helicase in the Escherichia coli replisome. It is a homohexameric enzyme that interacts with many other replisomal proteins and cofactors. It is usually loaded onto a single strand of DNA at origins of replication from its complex with its loading partner DnaC, then translocates in the 5' to 3' direction, unwinding duplex DNA in an NTP-driven process. Quaternary polymorphism has been described for the DnaB oligomer, a feature it has in common with some other hexameric helicases. In the present work, electron microscopy and in- depth rotational analysis studies of negatively stained specimens has allowed the establishment of conditions that govern the transition between the two different rotational symmetry states (C(3) and C(6)) of DnaB. It is shown: (a) that the pH value of the sample buffer, within the physiological range, dictates the quaternary organisation of the DnaB oligomer; (b) that the pH-induced transition is fully reversible; (c) that the type of adenine nucleotide complexed to DnaB, whether hydrolysable or not, does not affect its quaternary architecture; (d) that the DnaB.DnaC complex exists only as particles with C(3) symmetry; and (e) that DnaC interacts only with DnaB particles that have C(3) symmetry. Structural consequences of this quaternary polymorphism, as well as its functional implications for helicase activity, are discussed.

Adenosine Diphosphate↗
PubMed2000

Sequence-related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures.

RP4 TrbB, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4, is a member of the PulE protein superfamily involved in multicomponent machineries transporting macromolecules across the bacterial envelope. PulE-like proteins share several well conserved motifs, most notable a nucleoside triphosphate binding motif (P-loop). Helicobacter pylori HP0525 also belongs to the PulE superfamily and is encoded by the pathogenicity island cag, involved in the inflammatory response of infected gastric epithelial cells in mammals. The native molecular masses of TrbB and HP0525 as determined by gel filtration and glycerol gradient centrifugation suggested a homohexameric structure in the presence of ATP and Mg(2+). In the absence of nucleotides and bivalent cations, TrbB behaved as a tetramer whereas the hexameric state of HP0525 remained unaffected. Electron microscopy and image processing demonstrated that TrbB and HP0525 form ring-shaped complexes (diameter: 12 nm) with a central region (diameter: 3 nm) of low electron density when incubated in the presence of ATP and Mg(2+). However, the TrbB average image appeared to be more elliptical with strong twofold rotational symmetry whereas HP0525 complexes are regular hexagons. Six well defined triangle-shaped areas of high electron density were distinguishable in both cases. Covalent crosslinking of TrbB suggests that the hexameric ring is composed from a trimer of dimers, because only dimeric, tetrameric, and hexameric species were detectable. The toroidal structure of TrbB and HP0525 suggests that both proteins catalyze a repetitive process, most probably translocating a cognate substrate across the inner membrane.

Acid Anhydride Hydrolases↗
PubMed2000

The need for a shared database infrastructure: combining X-ray crystallography and electron microscopy.

Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases.

Crystallography, X-Ray↗
PubMed2000

Mapping and fuzzy classification of macromolecular images using self-organizing neural networks.

In this work the effectiveness of the fuzzy kohonen clustering network (FKCN) in the unsupervised classification of electron microscopic images of biological macromolecules is studied. The algorithm combines Kohonen's self-organizing feature maps (SOFM) and Fuzzy c-means (FCM) in order to obtain a powerful clustering technique with the best properties inherited from both. Exploratory data analysis using SOFM is also presented as a step previous to final clustering. Two different data sets obtained from the G40P helicase from B. Subtilis bacteriophage SPP1 have been used for testing the proposed method, one composed of 2458 rotational power spectra of individual images and the other composed by 338 images from the same macromolecule. Results of FKCN are compared with self-organizing feature maps (SOFM) and manual classification. Experimental results prove that this new technique is suitable for working with large, high-dimensional and noisy data sets and, thus, it is proposed to be used as a classification tool in electron microscopy.

Algorithms↗
PubMed2000

Large T-antigen double hexamers imaged at the simian virus 40 origin of replication.

The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg(2+) and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag-DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.

Antigens, Viral, Tumor↗
PubMed1999

The three-dimensional structure of a DNA translocating machine at 10 A resolution.

BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.

Bacillus Phages↗
PubMed1999

Organising multi-dimensional biological image information: the BioImage Database.

Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www. bioimage.org and http://www-embl.bioimage.org

Bacterial Proteins↗
PubMed1998

Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P).

The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication. The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate. We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein. Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views. Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture. These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole. A clear structural handedness has also been detected in some of these forms. An analysis of these quaternary states and a model for the structural organization of G40P are presented.

Bacillus subtilis↗
PubMed1998

Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.

BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity. RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein. CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

Bacterial Proteins↗