1,085 research outputs found

    The RCSB Protein Data Bank: redesigned web site and web services

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    The RCSB Protein Data Bank (RCSB PDB) web site (http://www.pdb.org) has been redesigned to increase usability and to cater to a larger and more diverse user base. This article describes key enhancements and new features that fall into the following categories: (i) query and analysis tools for chemical structure searching, query refinement, tabulation and export of query results; (ii) web site customization and new structure alerts; (iii) pair-wise and representative protein structure alignments; (iv) visualization of large assemblies; (v) integration of structural data with the open access literature and binding affinity data; and (vi) web services and web widgets to facilitate integration of PDB data and tools with other resources. These improvements enable a range of new possibilities to analyze and understand structure data. The next generation of the RCSB PDB web site, as described here, provides a rich resource for research and education

    Dimorphite-DL and biotite-tools, two open source programs for the acceleration of structure-based drug design

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    Computer-aided drug design has seen a proliferation of tools that allow the manipulation of small molecule and macromolecular structures in increasingly high-throughput settings. Molecular dynamics simulations, small molecule docking software, and visualization tools allow researchers to rapidly identify drug candidates and narrow the list of compounds that experimentalists must consider for further testing. Any gap in automating computer-aided drug design thus delays potentially life-saving discoveries. Here we present two open-source programs we developed to address challenges facing both protein and ligand preparation. Dimorphite-DL is a lightweight python program that predicts protonation states of small molecules using an empirical approach to ensure accurate docking and modelling calculations. The presence or absence of a hydrogen atom often determines whether a given ligand will bind a protein of interest. Biotite-tools is a python package that provides several popular statistical functions for analyzing molecular dynamics simulations in an easy-to-use way. Conformational fluctuation is complex, and it can be challenging to extract insight from what is essentially a “protein movie.” As such, simulation analysis has largely been restricted to those with backgrounds in computation, limiting the scope of such a powerful tool. Biotite-tools aims to accelerate the efforts of those already working with molecular dynamics and make analysis more accessible to experimentalists

    ANALYTICAL CONSIDERATIONS AND METHODS FOR COMPREHENSIVE ANALYSIS OF BACTERIAL PHOSPHOLIPIDOMICS USING HILIC-MS/MS

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    Omics technologies have rapidly evolved over the last half century through vast improvements in efficient extraction methodologies, advances in instrumentation for data collection, and a wide assortment of informatics tools to help deconvolute sample data sets. However, there are still untapped pools of molecules that warrant further analytical attention. As the frontline defense of the cell against exterior influences, the phospholipid membrane is key in structure, defense, and signaling, but current omics studies are only just now catching up to the potential hidden within cellular lipid profiles. Examination of shifts in phospholipid speciation and character could provide researchers with a wealth of information about how a cell attempts to adapt and survive when faced with adverse conditions. Application of such information could be valuable to the production of industrially relevant specialized bacterial strains, capable of processing large amounts of waste or feedstock as an affordable and renewable method. Using a flexible lipid extraction method using methyl tert-butyl ether (MTBE) combined with nanoscale hydrophilic interaction chromatography (HILIC) and nano-electrospray (nESI) tandem mass spectrometry (MS/MS), shifts in the lipidomes of several bacteria under consideration as industrial workhorses were investigated under variable growth conditions induced by introduction of toxic chemicals. Specific lipidome shifts were linked to specific growth conditions, which could lead to the production of bacterial strains designed to survive rough environments through genetic modification of phospholipid production

    The role of retromer in adipogenesis

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    Endocytosis is the process in which a cell engulfs extracellular cargo by creating invaginations within its plasma membrane. The cargo that has entered the cell enters an endosome and then is delivered to either the trans-Golgi network for recycling to the plasma membrane or to the lysosome for its degradation (Trousdale & Kim, 2015). Retromer is a peripheral membrane protein complex that plays a key role in sorting of these cargo molecules (Collins, 2008). More specifically, retromer deliver cargo from the endosome to the trans-Golgi Network, the process which is called retrograde transport of cargo molecules. Retromer dysfunction is strongly linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s Disease. However, recent Genome Wide Association Studies suggest that a mutation in retromer subunit VPS26a, has been linked to Type II Diabetes (Trousdale & Kim, 2015). A 2016 study published in The Faseb Journal attempts to characterize the role of retromer in adipocyte differentiation and insulin-stimulated uptake of glucose through transporter GLUT4 (Yang et al., 2016). The aim of this study is to further investigate the role of retromer in adipogenesis and to determine whether retromer plays a role at the transcriptional level or translational level. In this study, retromer’s VPS35 subunit was knocked down in four mouse 3T3-L1 fibroblast cell lines using the CRISPR-Cas9 approach. These cell lines were differentiated into mature adipocytes and analyzed by Oil-Red O staining, Western Blotting and quantitative PCR. The knockdown of retromer produced varying effects on adipocyte differentiation. In two of the knockdown cell lines, adipocyte differentiation was downregulated whereas adipocyte differentiation was upregulated in the other two cell lines. Although the results from Oil-Red O staining and Western Blot analyses complemented each other, results obtained from qPCR were not as straightforward and further analysis is needed to fully comprehend how retromer acts at the transcriptional level of cell differentiation. Based on the results of this study, retromer is involved in adipogenesis at both the transcriptional and translational level, however it’s mechanism of action remains unclear as both cases of impaired differentiation and upregulated differentiation were observed. Further studies are necessary to determine retromer’s exact role in adipogenesis

    Expanding the Known DNA-binding Specificity of Homeodomains for Utility in Customizable Sequence-specific Nucleases: A Dissertation

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    Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes

    Novel concepts for lipid identification from shotgun mass spectra using a customized query language

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    Lipids are the main component of semipermeable cell membranes and linked to several important physiological processes. Shotgun lipidomics relies on the direct infusion of total lipid extracts from cells, tissues or organisms into the mass spectrometer and is a powerful tool to elucidate their molecular composition. Despite the technical advances in modern mass spectrometry the currently available software underperforms in several aspects of the lipidomics pipeline. This thesis addresses these issues by presenting a new concept for lipid identification using a customized query language for mass spectra in combination with efficient spectra alignment algorithms which are implemented in the open source kit “LipidXplorer”

    Connectable Components for Protein Design

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    Protein design requires reusable, trustworthy, and connectable parts in order to scale to complex challenges. The recent explosion of protein structures stored within the Protein Data Bank provides a wealth of small motifs we can harvest, but we still lack tools to combine them into larger proteins. Here I explore two approaches for connecting reusable protein components on two different length scales. On the atomic scale, I build an interactive search engine for connecting chemical fragments together. Protein fragments built using this search engine recapitulate native-like protein assemblies that can be integrated into existing protein scaffolds using backbone search engines such as MaDCaT. On the protein domain scale, I quantitatively dissect structural variations in two-component systems in order to extract general principles for engineering interfacial flexibility between modular four-helix bundles. These bundles exhibit large scissoring motions where helices move towards or away from the bundle axis and these motions propagate across domain boundaries. Together, these two approaches form the beginnings of a multiscale methodology for connecting reusable protein fragments where there is a constant interplay and feedback between design of atomic structure, secondary structure, and tertiary structure. Rapid iteration, visualization, and search glue these diverse length scales together into a cohesive whole

    Geosensors to Support Crop Production: Current Applications and User Requirements

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    Sensor technology, which benefits from high temporal measuring resolution, real-time data transfer and high spatial resolution of sensor data that shows in-field variations, has the potential to provide added value for crop production. The present paper explores how sensors and sensor networks have been utilised in the crop production process and what their added-value and the main bottlenecks are from the perspective of users. The focus is on sensor based applications and on requirements that users pose for them. Literature and two use cases were reviewed and applications were classified according to the crop production process: sensing of growth conditions, fertilising, irrigation, plant protection, harvesting and fleet control. The potential of sensor technology was widely acknowledged along the crop production chain. Users of the sensors require easy-to-use and reliable applications that are actionable in crop production at reasonable costs. The challenges are to develop sensor technology, data interoperability and management tools as well as data and measurement services in a way that requirements can be met, and potential benefits and added value can be realized in the farms in terms of higher yields, improved quality of yields, decreased input costs and production risks, and less work time and load

    Strategic and practical guidelines for successful structured illumination microscopy

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    Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d
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