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.
Structural models; 3D data sets; Multi-platform; Modeling
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.
X-ray microscopy; full-field; tomography; XANES imaging
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.
electron microscopy; image processing workflow; distributed processing; micrograph alignment; fiducial markers
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.
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).
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.
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
Supplementary information: Additional information and a vignette are included in the beadarray package.
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.
molecular replacement; small-angle X-ray scattering (SAXS); wide-angle X-ray scattering (WAXS); molecular envelopes; heavy-atom location
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.
confidence intervals; fitting to low-resolution densities; electron microscopy
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.
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
Availability: XiP is open source, freely available under the Lesser General
Public License (LGPL) and can be downloaded from http://xip.hgc.jp.
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/.
stereochemical libraries; refinement; conformation-dependent library
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.
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.
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.
Brain research; Grid Computing; Workflows
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.
cryoelectron microscopy; cryoEM; semi-automatic data acquisition; electron microscope; CCD camera; scripting interface
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.
Synchrotron; modular detector; high frame rate detector; SAXS; WAXS; co-doped CsI
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.
time resolution; small-angle X-ray scattering (SAXS); shape reconstruction; biomolecules; biomolecular folding
Digitisation programmes in many institutes frequently involve disparate and irregular funding, diverse selection criteria and scope, with different members of staff managing and operating the processes. These factors have influenced the decision at the Royal Botanic Garden Edinburgh to develop an integrated workflow for the digitisation of herbarium specimens which is modular and scalable to enable a single overall workflow to be used for all digitisation projects. This integrated workflow is comprised of three principal elements: a specimen workflow, a data workflow and an image workflow.
The specimen workflow is strongly linked to curatorial processes which will impact on the prioritisation, selection and preparation of the specimens. The importance of including a conservation element within the digitisation workflow is highlighted. The data workflow includes the concept of three main categories of collection data: label data, curatorial data and supplementary data. It is shown that each category of data has its own properties which influence the timing of data capture within the workflow. Development of software has been carried out for the rapid capture of curatorial data, and optical character recognition (OCR) software is being used to increase the efficiency of capturing label data and supplementary data. The large number and size of the images has necessitated the inclusion of automated systems within the image workflow.
Large-scale digitisation; curation; data entry; image capture
Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.
Vesicular stomatitis virus (VSV) enters cells via endocytosis. At low pH, its unique glycoprotein G catalyzes fusion between viral and endosomal membranes by going from a pre-fusion trimeric conformation to a post-fusion trimeric conformation. There is a lack of information about G structural intermediates during the transition, in particular topological issues concerning the transition pathway, as the structural rearrangement cannot occur without breaking the threefold symmetry. Using several biophysical techniques, we analyzed the structure of a soluble form of G ectodomain at several pH values to follow the structural transition. We identified flexible monomeric intermediates that explore a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when the pH decreases. We propose that these monomers, also detected at the viral surface by electron microscopy, are intermediates during the fusion-associated conformational change and thus that VSV G ectodomain fully dissociates at the viral surface during the structural transition. This paper is an invitation to reconsider some data obtained on class I viral fusion glycoproteins (like Paramyxovirus F, influenza HA, HIV gp41) as for these proteins the topology of the conformational change is similar to that of VSV G.
The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development.
In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion.
Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.
Bioinformatic analyses typically proceed as chains of data-processing tasks. A pipeline, or 'workflow', is a well-defined protocol, with a specific structure defined by the topology of data-flow interdependencies, and a particular functionality arising from the data transformations applied at each step. In computer science, the dataflow programming (DFP) paradigm defines software systems constructed in this manner, as networks of message-passing components. Thus, bioinformatic workflows can be naturally mapped onto DFP concepts.
To enable the flexible creation and execution of bioinformatics dataflows, we have written a modular framework for parallel pipelines in Python ('PaPy'). A PaPy workflow is created from re-usable components connected by data-pipes into a directed acyclic graph, which together define nested higher-order map functions. The successive functional transformations of input data are evaluated on flexibly pooled compute resources, either local or remote. Input items are processed in batches of adjustable size, all flowing one to tune the trade-off between parallelism and lazy-evaluation (memory consumption). An add-on module ('NuBio') facilitates the creation of bioinformatics workflows by providing domain specific data-containers (e.g., for biomolecular sequences, alignments, structures) and functionality (e.g., to parse/write standard file formats).
PaPy offers a modular framework for the creation and deployment of parallel and distributed data-processing workflows. Pipelines derive their functionality from user-written, data-coupled components, so PaPy also can be viewed as a lightweight toolkit for extensible, flow-based bioinformatics data-processing. The simplicity and flexibility of distributed PaPy pipelines may help users bridge the gap between traditional desktop/workstation and grid computing. PaPy is freely distributed as open-source Python code at http://muralab.org/PaPy, and includes extensive documentation and annotated usage examples.
The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.
To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.
Endotoxin; Lipopolysaccharide; Antimicrobial peptide; Sepsis; Granulysin
Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.
Scanning transmission electron microscopy; STEM; electron tomography; Nanogold; Undecagold; labeling; protein