3DBIONOTES v2.0: a web server for the automatic annotation of macromolecular structures.

MOTIVATION: Complementing structural information with biochemical and biomedical annotations is a powerful approach to explore the biological function of macromolecular complexes. However, currently the compilation of annotations and structural data is a feature only available for those structures that have been released as entries to the Protein Data Bank. RESULTS: To help researchers in assessing the consistency between structures and biological annotations for structural models not deposited in databases, we present 3DBIONOTES v2.0, a web application designed for the automatic annotation of biochemical and biomedical information onto macromolecular structural models determined by any experimental or computational technique. AVAILABILITY AND IMPLEMENTATION: The web server is available at http://3dbionotes-ws.cnb.csic.es. CONTACT: jsegura@cnb.csic.es. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Local analysis of strains and rotations for macromolecular electron microscopy maps.

Macromolecular complexes perform their physiological functions by local rearrangements of their constituents and biochemically interacting with their reaction partners. These rearrangements may involve local rotations and the induction of local strains causing different mechanical efforts and stretches at the different areas of the protein. The analysis of these local deformations may reveal important insight into the way proteins perform their tasks. In this paper we introduce a method to perform this kind of local analysis using Electron Microscopy volumes in a fully objective and automatic manner. For doing so, we exploit the continuous nature of the result of an elastic image registration using B-splines as its basis functions. We show that the results obtained by the new automatic method are consistent with previous observations on these macromolecules.

3DBIONOTES: A unified, enriched and interactive view of macromolecular information.

With the advent of high throughput techniques like Next Generation Sequencing, the amount of biological information for genes and proteins is growing faster than ever. Structural information is also rapidly growing, especially in the cryo Electron Microscopy area. However, in many cases, the proteomic and genomic data are spread in multiple databases and with no simple connection to structural information. In this work we present a new web platform that integrates EMDB/PDB structures and UniProt sequences with different sources of protein annotations. The application provides an interactive interface linking sequence and structure, including EM maps, presenting the different sources of information at sequence and structural level. The web application is available at http://3dbionotes.cnb.csic.es.

A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs.

MOTIVATION: Structural information of macromolecular complexes provides key insights into the way they carry out their biological functions. Achieving high-resolution structural details with electron microscopy requires the identification of a large number (up to hundreds of thousands) of single particles from electron micrographs, which is a laborious task if it has to be manually done and constitutes a hurdle towards high-throughput. Automatic particle selection in micrographs is far from being settled and new and more robust algorithms are required to reduce the number of false positives and false negatives. RESULTS: In this article, we introduce an automatic particle picker that learns from the user the kind of particles he is interested in. Particle candidates are quickly and robustly classified as particles or non-particles. A number of new discriminative shape-related features as well as some statistical description of the image grey intensities are used to train two support vector machine classifiers. Experimental results demonstrate that the proposed method: (i) has a considerably low computational complexity and (ii) provides results better or comparable with previously reported methods at a fraction of their computing time. AVAILABILITY: The algorithm is fully implemented in the open-source Xmipp package and downloadable from http://xmipp.cnb.csic.es.

Particle quality assessment and sorting for automatic and semiautomatic particle-picking techniques.

Three-dimensional reconstruction of biological specimens using electron microscopy by single particle methodologies requires the identification and extraction of the imaged particles from the acquired micrographs. Automatic and semiautomatic particle selection approaches can localize these particles, minimizing the user interaction, but at the cost of selecting a non-negligible number of incorrect particles, which can corrupt the final three-dimensional reconstruction. In this work, we present a novel particle quality assessment and sorting method that can separate most erroneously picked particles from correct ones. The proposed method is based on multivariate statistical analysis of a particle set that has been picked previously using any automatic or manual approach. The new method uses different sets of particle descriptors, which are morphology-based, histogram-based and signal to noise analysis based. We have tested our proposed algorithm with experimental data obtaining very satisfactory results. The algorithm is freely available as a part of the Xmipp 3.0 package [http://xmipp.cnb.csic.es].

Automatic particle selection from electron micrographs using machine learning techniques.

The 3D reconstruction of biological specimens using Electron Microscopy is currently capable of achieving subnanometer resolution. Unfortunately, this goal requires gathering tens of thousands of projection images that are frequently selected manually from micrographs. In this paper we introduce a new automatic particle selection that learns from the user which particles are of interest. The training phase is semi-supervised so that the user can correct the algorithm during picking and specifically identify incorrectly picked particles. By treating such errors specially, the algorithm attempts to minimize the number of false positives. We show that our algorithm is able to produce datasets with fewer wrongly selected particles than previously reported methods. Another advantage is that we avoid the need for an initial reference volume from which to generate picking projections by instead learning which particles to pick from the user. This package has been made publicly available in the open-source package Xmipp.

Exploiting desktop supercomputing for three-dimensional electron microscopy reconstructions using ART with blobs.

Three-dimensional electron microscopy allows direct visualization of biological macromolecules close to their native state. The high impact of this technique in the structural biology field is highly correlated with the development of new image processing algorithms. In order to achieve subnanometer resolution, the size and number of images involved in a three-dimensional reconstruction increase and so do computer requirements. New chips integrating multiple processors are hitting the market at a reduced cost. This high-integration, low-cost trend has just begun and is expected to bring real supercomputers to our laboratory desktops in the coming years. This paper proposes a parallel implementation of a computation-intensive algorithm for three-dimensional reconstruction, ART, that takes advantage of the computational power in modern multicore platforms. ART is a sophisticated iterative reconstruction algorithm that has turned out to be well suited for the conditions found in three-dimensional electron microscopy. In view of the performance obtained in this work, these modern platforms are expected to play an important role to face the future challenges in three-dimensional electron microscopy.

A novel neural network technique for analysis and classification of EM single-particle images.

We propose a novel self-organizing neural network for the unsupervised classification of electron microscopy (EM) images of biological macromolecules. The radical novelty of the algorithm lies in its rigorous mathematical formulation that, starting from a large set of possibly very noisy input data, finds a set of "representative" data items, organized onto an ordered output map, such that the probability density of this set of representative items resembles at its possible best the probability density of the input data. In a way, it summarizes large amounts of information into a concise description that rigorously keeps the basic pattern of the input data distribution. In this application to the field of three-dimensional EM of single particles, two different data sets have been used; one comprised 2458 rotational power spectra of individual negative stain images of the G40P helicase of Bacillus subtilis bacteriophage SPP1, and the other contained 2822 cryoelectron images of SV40 large T-antigen. Our experimental results prove that this technique is indeed very successful, providing the user with the capability of exploring complex patterns in a succinct, informative, and objective manner. The above facts, together with the consideration that the integration of this new algorithm with commonly used software packages is immediate, prompt us to propose it as a valuable new tool in the analysis of large collections of noisy data.

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.

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.

The RepA protein of plasmid RSF1010 is a replicative DNA helicase.

The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (Röleke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5' --> 3' polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.

Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel.

The large T antigen of simian virus 40 (SV40) is a multifunctional regulatory protein, responsible for both the control of viral infection and the required alterations of cellular processes. T antigen is the only viral protein required for viral DNA replication. It binds specifically to the viral origin and as a helicase unwinds the SV40 DNA bidirectionally. The functional complex is a double hexameric oligomer. In the absence of DNA, but in the presence of ATP or a non-hydrolyzable analog, T antigen assembles into hexamers, which are active as a helicase when a partially single-stranded (3') entry site exists on the substrate. We have used negative staining electron microscopy, single particle image processing and three-dimensional reconstruction with a new algebraic reconstruction techniques (ART) algorithm to study the structure of these hexameric particles in the presence of different nucleotide cofactors (ATP, ADP, and the non-hydrolyzable analogs ATPgammaS and AMP-PNP). In every case a strong 6-fold structure was found, with the six density maxima arranged in a ring-like particle around a channel, and a well-defined vorticity. Because these structural features have recently been found in other prokaryotic helicases, they seem to be strongly related to the activity of the protein, which suggests a general functional model conserved through evolution.

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.

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.