Recent developments in software for the automation of crystallographic macromolecular structure determination

The automation of macromolecular structure determination by X-ray crystallography has long been a goal for many researchers. Recently, there have been improvements in the underlying algorithms, some of which have been implemented in software packages that deal with multiple stages of the structure determination process. These first steps towards complete automation have made X-ray crystallography more efficient.     

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

An Infrastructure for Integrated Automation System Implementation

Evolution of advanced manufacturing technologies and the new manufacturing paradigm has enriched the computer integrated manufacturing (CIM) methodology. The new advances have put more demands for CIM integration technology and associated supporting tools. One of these demands is to provide CIM systems with better software architecture, more flexible integration mechanisms, and powerful support platforms. In this paper, we present an integrating infrastructure for CIM implementation in manufacturing enterprises to form an integrated automation system. A research prototype of an integrating infrastructure has been developed for the development, integration, and operation of integrated CIM system. It is based on the client/server structure and employs object-oriented and agent technology. System openness, scalability, and maintenance are ensured by conforming to international standards and by using effective system design software and management tools.     

phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta

The combination of algorithms from the structure-modeling field with those of crystallographic structure determination can broaden the range of templates that are useful for structure determination by the method of molecular replacement. Automated tools in phenix.mr_rosetta simplify the application of these combined approaches by integrating Phenix crystallographic algorithms and Rosetta structure-modeling algorithms and by systematically generating and evaluating models with a combination of these methods. The phenix.mr_rosetta algorithms can be used to automatically determine challenging structures. The approaches used in phenix.mr_rosetta are described along with examples that show roles that structure-modeling can play in molecular replacement.     

Automation of random conical tilt and orthogonal tilt data collection using feature-based correlation

Visualization by electron microscopy has provided many insights into the composition, quaternary structure, and mechanism of macromolecular assemblies. By preserving samples in stain or vitreous ice it is possible to image them as discrete particles, and from these images generate three-dimensional structures. This ‘single-particle’ approach suffers from two major shortcomings; it requires an initial model to reconstitute 2D data into a 3D volume, and it often fails when faced with conformational variability. Random conical tilt (RCT) and orthogonal tilt (OTR) are methods developed to overcome these problems, but the data collection required, particularly for vitreous ice specimens, is difficult and tedious. In this paper, we present an automated approach to RCT/OTR data collection that removes the burden of manual collection and offers higher quality and throughput than is otherwise possible. We show example datasets collected under stain and cryo conditions and provide statistics related to the efficiency and robustness of the process. Furthermore, we describe the new algorithms that make this method possible, which include new calibrations, improved targeting and feature-based tracking.     

Corrim-based alignment for improved speed in single-particle image processing

The technique of single-particle electron cryomicroscopy is currently making possible the 3D structure determination of large macromolecular complexes at constantly increasing levels of resolution. Work at resolution now attainable requires many thousands of individual images to be processed computationally. The most time-consuming step of the image-processing procedure is usually the iterative alignment of individual particle images against a set of reference images derived from a preliminary 3-D structure. We have developed an improved multireference alignment procedure based on interpolated cross-correlation images (corrims) that results in an approximately 8-fold acceleration of the iterative alignment steps. These corrims can be used to restrict the number of image-alignment calculations by narrowing down the set of reference images. Another improvement in alignment speed has been achieved by optimising the software and its implementation on many parallel processors. This new corrim-based refinement has been found to work well with two different alignment algorithms, the commonly used "fast alignment by separate translational/rotational searches" and "exhaustive alignment by polar coordinates."     

Biskit--a software platform for structural bioinformatics

Biskit is a modular, object-oriented python library that provides intuitive classes for many typical tasks of structural bioinformatics research. It facilitates the manipulation and analysis of macromolecular structures, protein complexes and molecular dynamics trajectories. At the same time, Biskit offers a software platform for the rapid integration of external programs and new algorithms into complex structural bioinformatics workflows. Calculations are thus often delegated to established programs like Xplor, Amber, Hex, Prosa, Hmmer and Modeller; interfaces to further software can be easily added. Moreover, Biskit simplifies the parallelization of time consuming calculations via PVM (Parallel Virtual Machine). AVAILABILITY: The latest snapshot of Biskit, documentation and examples are freely available under the GNU General Public License at http://biskit.sf.net (alternate url http://biskit.pasteur.fr).     

Macromolecular recognition

Computational methods are being developed both to detect the binding surfaces of individual macromolecules and to predict the structure of binary macromolecular complexes. Speeding up and refining this process has required work on search algorithms, molecular representations and interaction potentials. Although backbone flexibility and solvent effects continue to pose problems, encouraging results have been obtained for both protein-protein and protein-DNA complexes.     

Applications of the polymerase chain reaction to genome analysis

The objectives of the Human Genome Project are to create high-resolution genetic and physical maps, and ultimately to determine the complete nucleotide sequence of the human genome. The result of this initiative will be to localize the estimated 50,000-100,000 human genes, and acquire information that will enable development of a better understanding of the relationship between genome structure and function. To achieve these goals, new methodologies that provide more rapid, efficient, and cost effective means of genomic analysis will be required. From both conceptual and practical perspectives, the polymerase chain reaction (PCR) represents a fundamental technology for genome mapping and sequencing. The availability of PCR has allowed definition of a technically credible form that the final composite map of the human genome will take, as described in the sequence-tagged site proposal. Moreover, applications of PCR have provided efficient approaches for identifying, isolating, mapping, and sequencing DNA, many of which are amenable to automation. The versatility and power provided by PCR have encouraged its involvement in almost every aspect of human genome research, with new applications of PCR being developed on a continual basis.     

Efficient implementation of a filtered back-projection algorithm using a voxel-by-voxel approach

The large amount of image data necessary for high-resolution 3D reconstruction of macromolecular assemblies leads to significant increases in the computational time. One of the most time consuming operations is 3D density map reconstruction, and software optimization can greatly reduce the time required for any given structural study. The majority of algorithms proposed for improving the computational effectiveness of a 3D reconstruction are based on a ray-by-ray projection of each image into the reconstructed volume. In this paper, we propose a novel fast implementation of the "filtered back-projection" algorithm based on a voxel-by-voxel principle. Our version of this implementation has been exhaustively tested using both model and real data. We compared 3D reconstructions obtained by the new approach with results obtained by the filtered Back-Projections algorithm and the Fourier-Bessel algorithm commonly used for reconstructing icosahedral viruses. These computational experiments demonstrate the robustness, reliability, and efficiency of this approach.     

GRASS: a server for the graphical representation and analysis of structures

GRASS (Graphical Representation and Analysis of Structures Server), a new web-based server, is described. GRASS exploits many of the features of the GRASP program and is designed to provide interactive molecular graphics and quantitative analysis tools with a simple interface over the World-Wide Web. Using GRASS, it is now possible to view many surface features of biological macromolecules on either standard workstations used in macromolecular analysis or personal computers. The result is a World-Wide Web-based, platform-independent, easily used tool for macromolecular visualization and structure analysis.     

Distant plant homologues: don't throw out the baby

Plants and metazoans share many similarities in terms of conserved proteins. Antibodies have been used extensively to detect remote homologues, many of which are yet to be identified conclusively. Genome sequencing and the creation of novel sequence or structure comparison programs have assisted greatly in the identification of distant protein homologues. The continuing development of new software algorithms and the combining of bioinformatics with proteomics offer hope that remaining homologues will be soon identified.     

An efficient medical database system implementation with its tool and data structure consideration

The development of a new system for medical database application running on minicomputer under MUMPS system is described. Two kinds of data representation in global structure are briefly reviewed. The use of a subject oriented multi-dimensional data structure greatly simplifies database design and facilitates data manipulation, organization, selective retrieval and software development. It is concluded that the program generator approach can provide the flexibility necessary for various applications and future growth of computerized medical record system. The final system has been proven effective in practical operation. The future extension concerns the introduction of multi-microprocessor structure and logic representation is presented.     

SoFAR: software for fully automatic evaluation of real-time PCR data

Quantitative real-time PCR has proven to be an extremely useful technique in life sciences for many applications. Although a lot of attention has been paid to the optimization of the assay conditions, the analysis of the data acquired is often done with software tools that do not make optimum use of the information provided by the data. Particularly, this is the case for high-throughput analysis, which requires a careful characterization and interpretation of the complete data by suitable software. Here we present a software solution for the robust, reliable, accurate, and fast evaluation of real-time PCR data, called SoFAR. The software automatically evaluates the data acquired with the LightCycler system. It applies new algorithms for an adaptive background correction of signal trends, the calculation of the effective signal noise, the automated identification of the exponential phases, the adaptive smoothing of the raw data, and the correction of melting curve data. Finally, it provides information regarding the validity of the results obtained. The SoFAR software minimizes the time required for evaluation and increases the accuracy and reliability of the results. The software is available upon request.