EMAN: semiautomated software for high-resolution single-particle reconstructions

We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.     

De novo identification of binding sequences for antibody replacement molecules

A new in silico method has been developed that automatically identifies peptide sequences that can bind to targets of known three‐dimensional structure. The method is potentially faster and more economical than traditional methods of raising antibodies by means of hybridomas or biopanning technology. The current algorithm creates an initial peptide library that is either completely random or that is constrained by the user. This library represents only a small fraction of possible sequence space and the peptides are created with a specified torsional geometry. The library is used as input to any number of available molecular docking programs and the library is docked and scored. The final rank ordering is then used to create a new library by constraining that library to the sequence conservation pattern deduced from the top N‐scoring peptides in the first round. Successive rounds of screening, scoring, and new library creation ultimately results in the system converging to a final solution set of peptides. To test the method, a family of novel peptides that can bind to, and inhibit the enzyme Deoxyribonuclease I has been discovered. The peptides inhibit the enzyme either alone or when placed into a protein backbone structure as has been confirmed experimentally. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.     

GUI programs for processing individual images in early stages of helical image reconstruction--for high-resolution structure analysis

A set of programs equipped with graphical user interface has been developed for processing individual images in early stages of the three-dimensional helical image reconstruction procedure. These programs can be used for initial screening of suitable image area, straightening the object image, determination of box parameters including the repeat distance, determination of the out-of-plane tilt and initial editing of the layer-line data. These tasks are difficult to automate and therefore very time-consuming. The programs, developed by adopting the concept of the layer-line indexing [Ultramicroscopy 84 (2000) 1-14], are effective for processing many images of filamentous molecular assemblies and especially tubular crystals having various helical classes. Using these programs, higher-resolution signals can be extracted more reliably and quickly, and the time required for processing each image can be reduced to 1/2-1/10. Here also presented is an overview on helical image reconstruction for high-resolution structure analysis.     

Trabecular bone densitometry using interactive image analysis

Interactive image analysis is a novel application of digital image processing to the densitometry of trabecular bone. Briefly, the exposed surfaces of bleached slices of bone are illuminated so that the trabecular tips are bright in relation to the interstices. The image is then captured by a TV camera and digitized with an interactive image analysis system. A grey scale is then chosen that differentiates the bone at the surface from the background. Finally, a computer program calculates the area fraction of bone within a user-specified box which, by Delesse's principle, is an estimate of the volume fraction of bone. The reproducibility of the technique (expressed as an average sd) is ± 1.5 vol. % and has surface-discriminating capabilities comparable to the traditional point-counting method but is faster and more precise. Most importantly, the technique permits biomechanical testing of the sample after its density has been determined.     

GCI: A network server for interactive 3D graphics

The Graphics Command Interpreter (GCI) is an independent server module that can be interfaced to any program that needs interactive three-dimensional (3D) graphics capabilities. The principal advantage of GCI is its simplicity. Only a limited set of powerful features have been implemented, including object management, global and local transformations, rotation, translation, clipping, scaling, viewport operations, window management, menu handling and picking.GCI and the master (client) program it serves run concurrently, communicating over a local or remote TCP/IP network. GCI sets up socket communication and provides a 3D graphics window and a terminal emulator for the master program. Communication between the two programs is via ASCII strings over standard I/O channels. The implied language for messages is very simple. GCI interprets messages from the master program and implements them as changes of graphical objects or as text messages to the user. GCI provides the user with facilities to manipulate the view of the displayed 3D objects interactively, independently of the master program, and to communicate mouse-controlled selection of menu items or 3D points as well as keyboard strings to the master program.The program is written in C and initially implemented using the Silicon Graphics GL graphics library. As the need to link special libraries to the master program is completely avoided, GCI can very easily be interfaced to existing programs written in any language and running on any operating system capable of TCP/IP communication. The program is freely available.     

MOL3D—A modular and interactive program for molecular modeling and conformational analysis: II. Extended modules

The new release of MOL3D, a molecular modeling program written in FORTRAN, contains not only enhanced graphic capabilities, but also an improved module for intermolecular calculations that allows rigid and flexible docking. Various interfaces have been added to some well-known and widely diffused programs, such as MM2, AMBER and MOPAC, and to the Cambridge Crystallographic Database. Finally a graph manager and a samples database have been added, which allow efficient searches with various requirements concerning structural templates, pharmacophoric three-dimensional (3D) constraints, and the field of biological activity, if any.     

Using digital image processing for counting whiteflies on soybean leaves

This paper presents a new system, based on digital image processing, to quantify whiteflies on soybean leaves. This approach allows counting to be fully automated, considerably speeding up the process in comparison with the manual approach. The proposed algorithm is capable of detecting and quantifying not only adult whiteflies, but also specimens in the nymph stage. A complete performance evaluation is presented, with emphasis on the conditions and situations for which the algorithm succeeds, and also on the circumstances that need further work. Although this proposal was entirely developed using soybean leaves, it can be easily extended to other kinds of crops with little or no changes in the algorithm. The system employs only widely used image processing operations, so it can be easily implemented in any image processing software package.     

Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.     

Development of a bacteriophage-based system for the selection of structured peptides

Short structured peptides can provide scaffolds for protease-resistant peptide therapeutics, serve as useful building blocks in biomedical and biotechnological applications, and shed light on the role of secondary structure elements in protein folding. It is well known that directed evolution is a powerful method for creating proteins and peptides with novel properties, and a system for the selection of short peptides based on structure from a randomized library would be an important advancement. In this study, phage particles monovalently displaying a short peptide and an N-terminal 6×His tag on their P3 coat protein were bound to nickel agarose resin and were subsequently challenged with a protease that specifically cleaves at a site within the peptide. The extent to which phage is proteolytically released from the resin was found to be dependent on the structural properties of the inserted peptide sequences. As proofs-of-concept, a structured peptide has been isolated from a pool of flexible peptides using a trypsin selection, and a flexible peptide has been isolated from a pool of structured peptides using a chymotrypsin selection. This selection system will be a strong technological platform for the creation of short peptides with interesting structural properties using directed evolution.     

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.     

Software development of the EMI head scanner.

A method has been developed to run the general purpose operating system RDOS on the same disc of the head scanner computer as is used for scanner software and data. This made it possible to develop additional software in high level programming language for image processing, based on original image data on the disc. All new images produced by the program are stored on the disc in the same format as the original images. This makes it possible to handle processed images exactly as the original ones and to do multiple operations. The following processing has been included in the program so far: subtraction, smoothing, density profiles, vertical reconstructions, magnification and labelling. A set of operator commands has been developed which are very similar to the ordinary commands for the scanner, which makes the program to appear being a direct extension of the standard scanner software.     

新版DELTA系统在植物分类学中的应用——以羊茅属研究为例

DELTA系统是针对分类学研究开发的综合性多功能软件包。新版DELTA系统在运行环境、程序、指令、界面、操作方式及中文字符处理等重要功能上比旧版本均有较大改进。作为国际分类学数据库数据交换标准,应用该系统可以规范分类学描述,对研究中所有常用性状进行识别和处理,自动生成各个研究对象分类学特征描述并对其进行详尽的相似性和差异性比较,对性状特征和分类单元进行注释和图像示例,将数据转换为数量分类学或分支分类学所需格式以生成树状图,自动生成检索表,建成基于本地和网络的交互式专家辅助鉴定系统等。DELTA系统作为植物分类学研究的常用手段和工具是非常适合及大有帮助的。     

jmzTab: A Java interface to the mzTab data standard

mzTab is the most recent standard format developed by the Proteomics Standards Initiative. mzTab is a flexible tab‐delimited file that can capture identification and quantification results coming from MS‐based proteomics and metabolomics approaches. We here present an open‐source Java application programming interface for mzTab called jmzTab. The software allows the efficient processing of mzTab files, providing read and write capabilities, and is designed to be embedded in other software packages. The second key feature of the jmzTab model is that it provides a flexible framework to maintain the logical integrity between the metadata and the table‐based sections in the mzTab files. In this article, as two example implementations, we also describe two stand‐alone tools that can be used to validate mzTab files and to convert PRIDE XML files to mzTab. The library is freely available at http://mztab.googlecode.com .     

酵母双杂交技术应用进展

酵母双杂交技术是鉴定蛋白互作最有效和最广泛的分子生物学技术。该技术能直接作用于活细胞,检测细胞内蛋白质互作,具有成本低、易操作、可达到全基因组水平、能进行品种间的互作鉴定等诸多优点。较之传统的检测方法有明显优势,已在越来越多的领域得到应用。对酵母双杂交的技术原理以及应用进行了综述,介绍了该技术在发现新蛋白质、探究蛋白质功能、建立基因组蛋白连锁图、研究人类DNA文库和筛选药物作用位点等方面的重要应用,以期为该技术的广泛应用提供参考。     

An automated program to find animals and crop photographs for individual recognition

Detailed data on individual animals are critical to ecological and evolutionary studies, but attaching identifying marks can alter individual fates and behavior leading to biases in parameter estimates and ethical issues. Individual-recognition software has been developed to assist in identifying many species from non-invasive photographic data. These programs utilize algorithms to find unique individual characteristics and compare images to a catalogue of known individuals. Currently, all applications for individual identification require manual processing to crop images so only the area of interest remains, or the area of interest must be manually delineated in each image. Thus, one of the main bottlenecks in processing data from photographic capture-recapture surveys is in cropping to an area of interest so that matching algorithms can identify the individual. Here, we describe the development and testing of an automated cropping program. The methods and techniques we describe are broadly applicable to any system where raw photos must be cropped down to a specific area of interest before pattern recognition software can be used for individual identification. We developed and tested the program for use with identification photos of wild giraffes.     

medplot: A Web Application for Dynamic Summary and Analysis of Longitudinal Medical Data Based on R

In biomedical studies the patients are often evaluated numerous times and a large number of variables are recorded at each time-point. Data entry and manipulation of longitudinal data can be performed using spreadsheet programs, which usually include some data plotting and analysis capabilities and are straightforward to use, but are not designed for the analyses of complex longitudinal data. Specialized statistical software offers more flexibility and capabilities, but first time users with biomedical background often find its use difficult. We developed medplot, an interactive web application that simplifies the exploration and analysis of longitudinal data. The application can be used to summarize, visualize and analyze data by researchers that are not familiar with statistical programs and whose knowledge of statistics is limited. The summary tools produce publication-ready tables and graphs. The analysis tools include features that are seldom available in spreadsheet software, such as correction for multiple testing, repeated measurement analyses and flexible non-linear modeling of the association of the numerical variables with the outcome. medplot is freely available and open source, it has an intuitive graphical user interface (GUI), it is accessible via the Internet and can be used within a web browser, without the need for installing and maintaining programs locally on the user’s computer. This paper describes the application and gives detailed examples describing how to use the application on real data from a clinical study including patients with early Lyme borreliosis.