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1.  Using Situs for the integration of multi-resolution structures 
Biophysical Reviews  2010;2(1):21-27.
Situs is a modular and widely used software package for the integration of biophysical data across the spatial resolution scales. It has been developed over the last decade with a focus on bridging the resolution gap between atomic structures, coarse-grained models, and volumetric data from low-resolution biophysical origins, such as electron microscopy, tomography, or small-angle scattering. Structural models can be created and refined with various flexible and rigid body docking strategies. The software consists of multiple, stand-alone programs for the format conversion, analysis, visualization, manipulation, and assembly of 3D data sets. The programs have been ported to numerous platforms in both serial and shared memory parallel architectures and can be combined in various ways for specific modeling applications. The modular design facilitates the updating of individual programs and the development of novel application workflows. This review provides an overview of the Situs package as it exists today with an emphasis on functionality and workflows supported by version 2.5.
Electronic supplementary material
The online version of this article (doi:10.1007/s12551-009-0026-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s12551-009-0026-3
PMCID: PMC2821521  PMID: 20174447
Structural models; 3D data sets; Multi-platform; Modeling
2.  TXM-Wizard: a program for advanced data collection and evaluation in full-field transmission X-ray microscopy 
Journal of Synchrotron Radiation  2012;19(Pt 2):281-287.
A suite of GUI programs written in MATLAB for advanced data collection and analysis of full-field transmission X-ray microscopy data including mosaic imaging, tomography and XANES imaging is presented.
Transmission X-ray microscopy (TXM) has been well recognized as a powerful tool for non-destructive investigation of the three-dimensional inner structure of a sample with spatial resolution down to a few tens of nanometers, especially when combined with synchrotron radiation sources. Recent developments of this technique have presented a need for new tools for both system control and data analysis. Here a software package developed in MATLAB for script command generation and analysis of TXM data is presented. The first toolkit, the script generator, allows automating complex experimental tasks which involve up to several thousand motor movements. The second package was designed to accomplish computationally intense tasks such as data processing of mosaic and mosaic tomography datasets; dual-energy contrast imaging, where data are recorded above and below a specific X-ray absorption edge; and TXM X-ray absorption near-edge structure imaging datasets. Furthermore, analytical and iterative tomography reconstruction algorithms were implemented. The compiled software package is freely available.
doi:10.1107/S0909049511049144
PMCID: PMC3284347  PMID: 22338691
X-ray microscopy; full-field; tomography; XANES imaging
3.  Automated Tracing of Filaments in 3D Electron Tomography Reconstructions using Sculptor and Situs 
Journal of structural biology  2012;178(2):121-128.
The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7 and Sculptor 2.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.
doi:10.1016/j.jsb.2012.03.001
PMCID: PMC3440181  PMID: 22433493
Tomograms; 3D analysis; filament detection; actin networks; denoising; segmentation
4.  Computational Resources for Cryo-Electron Tomography in Bsoft 
Journal of structural biology  2007;161(3):232-242.
The Bsoft package (Heymann and Belnap JSB 2007, 157, 3) has been enhanced by adding utilities for processing electron tomographic (ET) data; in particular, cryo-ET data characterized by low contrast and high noise. To handle the high computational load efficiently, a workflow was developed, based on the database-like parameter handling in Bsoft, aimed at minimizing user interaction and facilitating automation. To the same end, scripting elements distribute the processing among multiple processors on the same or different computers. The resolution of a tomogram depends on the precision of projection alignment, which is usually based on pinpointing fiducial markers (electron-dense gold particles). Alignment requires accurate specification of the tilt axis, and our protocol includes a procedure for determining it to adequate accuracy. Refinement of projection alignment provides information that allows assessment of its precision, as well as projection quality control. We implemented a reciprocal space algorithm that affords an alternative to back-projection or real space algorithms for calculating tomograms Resources are also included that allow resolution assessment by cross-validation (NLOO2D); denoising and interpretation; and the extraction, mutual alignment, and averaging of tomographic subvolumes.
doi:10.1016/j.jsb.2007.08.002
PMCID: PMC2409064  PMID: 17869539
electron microscopy; image processing workflow; distributed processing; micrograph alignment; fiducial markers
5.  Image processing for electron microscopy single-particle analysis using XMIPP 
Nature protocols  2008;3(6):977-990.
We describe a collection of standardized image processing protocols for electron microscopy single-particle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.
doi:10.1038/nprot.2008.62
PMCID: PMC2778070  PMID: 18536645
6.  FoXS: a web server for rapid computation and fitting of SAXS profiles 
Nucleic Acids Research  2010;38(Web Server issue):W540-W544.
Small angle X-ray scattering (SAXS) is an increasingly common technique for low-resolution structural characterization of molecules in solution. SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. SAXS profiles can contribute to many applications, such as comparing a conformation in solution with the corresponding X-ray structure, modeling a flexible or multi-modular protein, and assembling a macromolecular complex from its subunits. These applications require rapid computation of a SAXS profile from a molecular structure. FoXS (Fast X-Ray Scattering) is a rapid method for computing a SAXS profile of a given structure and for matching of the computed and experimental profiles. Here, we describe the interface and capabilities of the FoXS web server (http://salilab.org/foxs).
doi:10.1093/nar/gkq461
PMCID: PMC2896111  PMID: 20507903
7.  Contrast-Matched Small Angle X-ray Scattering from a Heavy Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin-Peptide Complex 
Journal of the American Chemical Society  2012;134(36):14686-14689.
The information content in one-dimensional solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique three-dimensional structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small and wide angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca2+ ions have been substituted by Pb2+, and a target peptide from myosin light chain kinase. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data.
doi:10.1021/ja306359z
PMCID: PMC3442789  PMID: 22908850
8.  Structure and flexibility within proteins as identified through small angle X-ray scattering 
General physiology and biophysics  2009;28(2):174-189.
Flexibility between domains of proteins is often critical for function. These motions and proteins with large scale flexibility in general are often not readily amenable to conventional structural analysis such as X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR) or electron microscopy. A common evolution of a crystallography project, once a high resolution structure has been determined, is to postulate possible sights of flexibility. Here we describe an analysis tool using relatively inexpensive small angle X-ray scattering (SAXS) measurements to identify flexibility and validate a constructed minimal ensemble of models, which represent highly populated conformations in solution. The resolution of these results is sufficient to address the questions being asked: what kinds of conformations do the domains sample in solution? In our rigid body modeling strategy BILBOMD, molecular dynamics (MD) simulations are used to explore conformational space. A common strategy is to perform the MD simulation on the domains connections at very high temperature, where the additional kinetic energy prevents the molecule from becoming trapped in a local minimum. The MD simulations provide an ensemble of molecular models from which a SAXS curve is calculated and compared to the experimental curve. A genetic algorithm is used to identify the minimal ensemble (minimal ensemble search, MES) required to best fit the experimental data. We demonstrate the use of MES in several model and in four experimental examples.
PMCID: PMC3773563  PMID: 19592714
Small angle X-ray scattering; Protein flexibility; Molecular dynamics; Rigid body modeling
9.  Galaxy for Core Facilities 
Galaxy is an open, web-based platform for data intensive biological research that enables non-bioinformaticians to create, run, tune, and share their own bioinformatic analyses. Galaxy can be used in core facilities to 1) automate analysis pipelines, 2) enable clients to see the details of how their results were produced, from raw inputs through finished products, 3) enable clients to explore, modify and reuse a facility's default analysis workflows, and 4) enable clients to develop their own workflows without having to learn a programming language, use a command line interface, learn Linux package management, or require extensive hand-holding from core facility staff. This talk will introduce Galaxy and highlight how core facilities can use it to empower their clients and reduce staff workload. This talk will also cover programmatic/scripting access via the Galaxy API, relevant Galaxy community resources, Galaxy's history and reproducibility features, and multiple Galaxy deployment options including cloud-based deployments and installing Galaxy on local computational resources.
PMCID: PMC3635312
10.  Workflow and Electronic Health Records in Small Medical Practices 
This paper analyzes the workflow and implementation of electronic health record (EHR) systems across different functions in small physician offices. We characterize the differences in the offices based on the levels of computerization in terms of workflow, sources of time delay, and barriers to using EHR systems to support the entire workflow. The study was based on a combination of questionnaires, interviews, in situ observations, and data collection efforts. This study was not intended to be a full-scale time-and-motion study with precise measurements but was intended to provide an overview of the potential sources of delays while performing office tasks. The study follows an interpretive model of case studies rather than a large-sample statistical survey of practices. To identify time-consuming tasks, workflow maps were created based on the aggregated data from the offices. The results from the study show that specialty physicians are more favorable toward adopting EHR systems than primary care physicians are. The barriers to adoption of EHR systems by primary care physicians can be attributed to the complex workflows that exist in primary care physician offices, leading to nonstandardized workflow structures and practices. Also, primary care physicians would benefit more from EHR systems if the systems could interact with external entities.
PMCID: PMC3329208  PMID: 22737096
11.  A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario 
Frontiers in Zoology  2013;10:44.
Background
In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples.
Results
We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system.
Conclusions
The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations.
doi:10.1186/1742-9994-10-44
PMCID: PMC3750762  PMID: 23915384
12.  Combining solution wide-angle X-ray scattering and crystallography: determination of molecular envelope and heavy-atom sites 
Journal of Applied Crystallography  2009;42(Pt 2):259-264.
Molecular envelopes determined from SAXS/WAXS solution scattering can be used to locate the heavy-atom sites in the crystallographic unit cell.
Solving the phase problem remains central to crystallographic structure determination. A six-dimensional search method of molecular replacement (FSEARCH) can be used to locate a low-resolution molecular envelope determined from small-angle X-ray scattering (SAXS) within the crystallographic unit cell. This method has now been applied using the higher-resolution envelope provided by combining SAXS and WAXS (wide-angle X-ray scattering) data. The method was tested on horse hemoglobin, using the most probable model selected from a set of a dozen bead models constructed from SAXS/WAXS data using the program GASBOR at 5 Å resolution (q max = 1.25 Å−1) to phase a set of single-crystal diffraction data. It was found that inclusion of WAXS data is essential for correctly locating the molecular envelope in the crystal unit cell, as well as for locating heavy-atom sites. An anomalous difference map was calculated using phases out to 8 Å resolution from the correctly positioned envelope; four distinct peaks at the 3.2σ level were identified, which agree well with the four iron sites of the known structure (Protein Data Bank code 1ns9). In contrast, no peaks could be found close to the iron sites if the molecular envelope was constructed using the data from SAXS alone (q max = 0.25 Å−1). The initial phases can be used as a starting point for a variety of phase-extension techniques, successful application of which will result in complete phasing of a crystallographic data set and determination of the internal structure of a macromolecule to atomic resolution. It is anticipated that the combination of FSEARCH and WAXS techniques will facilitate the initial structure determination of proteins and provide a good foundation for further structure refinement.
doi:10.1107/S0021889809003094
PMCID: PMC2677545  PMID: 19529837
molecular replacement; small-angle X-ray scattering (SAXS); wide-angle X-ray scattering (WAXS); molecular envelopes; heavy-atom location
13.  Confidence intervals for fitting of atomic models into low-resolution densities 
This paper describes procedures for obtaining confidence intervals for coordinate locations resulting from the fitting of atomic models into low-resolution densities.
The fitting of high-resolution structures into low-resolution densities obtained from techniques such as electron microscopy or small-angle X-ray scattering can yield powerful new insights. While several algorithms for achieving optimal fits have recently been developed, relatively little effort has been devoted to developing objective measures for judging the quality of the resulting fits, in particular with regard to the danger of overfitting. Here, a general method is presented for obtaining confidence intervals for atomic coordinates resulting from fitting of atomic resolution domain structures into low-resolution densities using well established statistical tools. It is demonstrated that the resulting confidence intervals are sufficiently accurate to allow meaningful statistical tests and to provide tools for detecting potential overfitting.
doi:10.1107/S0907444909012876
PMCID: PMC2703574  PMID: 19564688
confidence intervals; fitting to low-resolution densities; electron microscopy
14.  BASH: a tool for managing BeadArray spatial artefacts 
Bioinformatics  2008;24(24):2921-2922.
Summary: With their many replicates and their random layouts, Illumina BeadArrays provide greater scope fordetecting spatial artefacts than do other microarray technologies. They are also robust to artefact exclusion, yet there is a lack of tools that can perform these tasks for Illumina. We present BASH, a tool for this purpose. BASH adopts the concepts of Harshlight, but implements them in a manner that utilizes the unique characteristics of the Illumina technology. Using bead-level data, spatial artefacts of various kinds can thus be identified and excluded from further analyses.
Availability: The beadarray Bioconductor package (version 1.10 onwards), www.bioconductor.org
Contact: andy.lynch@cancer.org.uk
Supplementary information: Additional information and a vignette are included in the beadarray package.
doi:10.1093/bioinformatics/btn557
PMCID: PMC2639304  PMID: 18953044
15.  The Power of Correlative Microscopy: Multi-modal, Multi-scale, Multi-dimensional 
Correlative microscopy is a sophisticated approach that combines the capabilities of typically separate, but powerful microscopy platforms: often including, but not limited, to conventional light, confocal and super-resolution microscopy, atomic force microscopy, transmission and scanning electron microscopy, magnetic resonance imaging and micro/nanoCT (computed tomography). When targeting rare or specific events within large populations or tissues, correlative microscopy is increasingly being recognized as the method of choice. Furthermore, this multi-modal assimilation of technologies provides complementary and often unique information, such as internal and external spatial, structural, biochemical and biophysical details from the same targeted sample. The development of a continuous stream of cutting-edge applications, probes, preparation methodologies, hardware and software developments will enable realization of the full potential of correlative microscopy.
doi:10.1016/j.sbi.2011.06.010
PMCID: PMC3189301  PMID: 21782417
16.  A conformation-dependent stereochemical library improves crystallographic refinement even at atomic resolution 
A script was created to allow SHELXL to use the new CDL v.1.2 stereochemical library which defines the target values for main-chain bond lengths and angles as a function of the residue’s ϕ/ψ angles. Test refinements using this script show that the refinement behavior of structures at resolutions even better than 1 Å is substantially enhanced by the use of the new conformation-dependent ideal geometry paradigm.
To utilize a new conformation-dependent backbone-geometry library (CDL) in protein refinements at atomic resolution, a script was written that creates a restraint file for the SHELXL refinement program. It was found that the use of this library allows models to be created that have a substantially better fit to main-chain bond angles and lengths without degrading their fit to the X-ray data even at resolutions near 1 Å. For models at much higher resolution (∼0.7 Å), the refined model for parts adopting single well occupied positions is largely independent of the restraints used, but these structures still showed much smaller r.m.s.d. residuals when assessed with the CDL. Examination of the refinement tests across a wide resolution range from 2.4 to 0.65 Å revealed consistent behavior supporting the use of the CDL as a next-generation restraint library to improve refinement. CDL restraints can be generated using the service at http://pgd.science.oregonstate.edu/cdl_shelxl/.
doi:10.1107/S090744491102292X
PMCID: PMC3144852  PMID: 21795811
stereochemical libraries; refinement; conformation-dependent library
17.  IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure 
PLoS ONE  2012;7(1):e30249.
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
doi:10.1371/journal.pone.0030249
PMCID: PMC3265479  PMID: 22291925
18.  Unique Properties of Eukaryote-Type Actin and Profilin Horizontally Transferred to Cyanobacteria 
PLoS ONE  2012;7(1):e29926.
A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 µm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 µm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.
doi:10.1371/journal.pone.0029926
PMCID: PMC3254629  PMID: 22253827
19.  Structure of liposome encapsulating proteins characterized by X-ray scattering and shell-modeling 
Journal of Synchrotron Radiation  2013;20(Pt 6):869-874.
Wide-angle X-ray scattering data using a third-generation synchrotron radiation source are presented.
Lipid liposomes are promising drug delivery systems because they have superior curative effects owing to their high adaptability to a living body. Lipid liposomes encapsulating proteins were constructed and the structures examined using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). The liposomes were prepared by a sequential combination of natural swelling, ultrasonic dispersion, freeze-throw, extrusion and spin-filtration. The liposomes were composed of acidic glycosphingolipid (ganglioside), cholesterol and phospholipids. By using shell-modeling methods, the asymmetric bilayer structure of the liposome and the encapsulation efficiency of proteins were determined. As well as other analytical techniques, SR-SWAXS and shell-modeling methods are shown to be a powerful tool for characterizing in situ structures of lipid liposomes as an important candidate of drug delivery systems.
doi:10.1107/S0909049513020827
PMCID: PMC3795546  PMID: 24121330
solution X-ray scattering; liposome; DDS
20.  XiP: a computational environment to create, extend and share workflows 
Bioinformatics  2012;29(1):137-139.
XiP (eXtensible integrative Pipeline) is a flexible, editable and modular environment with a user-friendly interface that does not require previous advanced programming skills to run, construct and edit workflows. XiP allows the construction of workflows by linking components written in both R and Java, the analysis of high-throughput data in grid engine systems and also the development of customized pipelines that can be encapsulated in a package and distributed. XiP already comes with several ready-to-use pipeline flows for the most common genomic and transcriptomic analysis and ∼300 computational components.
Availability: XiP is open source, freely available under the Lesser General Public License (LGPL) and can be downloaded from http://xip.hgc.jp.
Contact: nagasaki@megabank.tohoku.ac.jp
doi:10.1093/bioinformatics/bts630
PMCID: PMC3530915  PMID: 23104885
21.  A Script Assisted Microscopy (SAM) Package to Improve Data Acquisition Rates on FEI Tecnai Electron Microscopes equipped with Gatan CCD Cameras 
Journal of structural biology  2008;164(1):166-169.
High throughput methods of data acquisition are advantageous for cryoelectron microscopy and single particle reconstruction as high-resolution structure determination requires thousands of particle images. We have developed a semi-automated data collection method that utilizes the scripting languages provided by FEI for their Tecnai User Interface (TUI) and by Gatan for their Digital Micrograph package. Our Script Assisted Microscopy (SAM) method allows for the selection of multiple locations within a low magnification, search mode, micrograph and for subsequent automated imaging of these locations at a higher exposure magnification. The SAM approach permits the user to retain control over the microscope, while streamlining the most repetitive steps of collecting and evaluating micrographs. With SAM, we have found an average of 1,000 micrographs can be collected per day on any grid type, either irregular homemade grids or prefabricated grids with regularly spaced holes. This rate of data collection represents a five-fold improvement over our manual collection rates. SAM provides an example of an individually tailored approach to data acquisition utilizing the scripting interfaces provided by the equipment manufacturers. The SAM method has proven valuable for determination of a subnanometer resolution cryoEM structure of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a 469kDa protein.
doi:10.1016/j.jsb.2008.05.011
PMCID: PMC2572106  PMID: 18621546
cryoelectron microscopy; cryoEM; semi-automatic data acquisition; electron microscope; CCD camera; scripting interface
22.  Reconstructing three-dimensional shape envelopes from time-resolved small-angle X-ray scattering data 
Journal of Applied Crystallography  2008;41(Pt 6):1046-1052.
The three-dimensional reconstruction program DAMMIN has been applied to time-resolved small-angle X-ray scattering data. The results are presented and their success in representing the molecules is assessed.
Modern computing power has made it possible to reconstruct low-resolution, three-dimensional shapes from solution small-angle X-ray scattering (SAXS) data on biomolecules without a priori knowledge of the structure. In conjunction with rapid mixing techniques, SAXS has been applied to time resolve conformational changes accompanying important biological processes, such as biomolecular folding. In response to the widespread interest in SAXS reconstructions, their value in conjunction with such time-resolved data has been examined. The group I intron from Tetrahymena thermophila and its P4–P6 subdomain are ideal model systems for investigation owing to extensive previous studies, including crystal structures. The goal of this paper is to assay the quality of reconstructions from time-resolved data given the sacrifice in signal-to-noise required to obtain sharp time resolution.
doi:10.1107/S0021889808028264
PMCID: PMC2648657  PMID: 19529835
time resolution; small-angle X-ray scattering (SAXS); shape reconstruction; biomolecules; biomolecular folding
23.  Sample Preparation Methods to Analyze DNA-Induced Structural Changes in Replication Protein A 
Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding protein (SSB) in eukaryotes, serves to protect ssDNA generated during these events and to recruit and organize other DNA-processing factors requiring access to ssDNA substrates. RPA engages ssDNA in distinct, progressive binding modes, which are thought to correspond to different functional states of the protein during the course of DNA processing. Structural characterization of these unique complexes has remained challenging, however, as RPA is a multi-domain protein characterized by a flexible, modular organization. Biophysical approaches that are well suited to probing time-varying architectures, such as NMR and small-angle X-ray and neutron scattering (SAXS/SANS), when integrated with computational methods, can provide critical insights into the architectural changes associated with RPA’s different DNA-binding modes. The success of these methods, however, is highly contingent upon the purity, homogeneity, and stability of the sample under study. Here we describe a basic protocol for characterizing and optimizing sample conditions for RPA/ssDNA complexes prior to study by SAXS and/or SANS.
doi:10.1007/978-1-62703-032-8_6
PMCID: PMC3713622  PMID: 22976179
Replication protein A; Single-stranded DNA-binding protein; Oligonucleotide-/oligosaccharide-binding fold; DNA processing; Protein modularity; Solubility screening; Small-angle X-ray scattering; Small-angle neutron scattering; Size-exclusion chromatography; Multi-angle light scattering
24.  Modular high frame rate detector for synchrotron applications 
The development of detectors often lags the development in X-ray sources. However, advanced detectors are critical for fully utilizing and exploiting the capabilities of the new bright sources. We report on the development of a modular high frame rate detector for synchrotron applications such as small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS). The detector consists of four modules, each providing an imaging area of 5×5 cm2 and capable of frame rates of 200 frames per second (fps) with full resolution, and 650 fps with smaller region of interest (ROI). Details of the detector design and experiments at synchrotron beamlines are discussed in the paper.
doi:10.1016/j.nima.2011.01.057
PMCID: PMC3150570  PMID: 21822342
Synchrotron; modular detector; high frame rate detector; SAXS; WAXS; co-doped CsI
25.  Accelerating Medical Research using the Swift Workflow System 
Both medical research and clinical practice are starting to involve large quantities of data and to require large-scale computation, as a result of the digitization of many areas of medicine. For example, in brain research – the domain that we consider here – a single research study may require the repeated processing, using computationally demanding and complex applications, of thousands of files corresponding to hundreds of functional MRI studies. Execution efficiency demands the use of parallel or distributed computing, but few medical researchers have the time or expertise to write the necessary parallel programs.
The Swift system addresses these concerns. A simple scripting language, SwiftScript, provides for the concise high-level specification of workflows that invoke various application programs on potentially large quantities of data. The Swift engine provides for the efficient execution of these workflows on sequential computers, parallel computers, and/or distributed grids that federate the computing resources of many sites. Last but not least, the Swift provenance catalog keeps track of all actions performed, addressing vital bookkeeping functions that so often cause difficulties in large computations.
To illustrate the use of Swift for medical research, we describe its use for the analysis of functional MRI data as part of a research project examining the neurological mechanisms of recovery from aphasia after stroke. We show how SwiftScript is used to encode an application workflow, and present performance results that demonstrate our ability to achieve significant speedups on both a local parallel computing cluster and multiple parallel clusters at distributed sites.
PMCID: PMC2676238  PMID: 17476063
Brain research; Grid Computing; Workflows

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