7,006 research outputs found

    Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

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    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

    SPEDEN: Reconstructing single particles from their diffraction patterns

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    Speden is a computer program that reconstructs the electron density of single particles from their x-ray diffraction patterns, using a single-particle adaptation of the Holographic Method in crystallography. (Szoke, A., Szoke, H., and Somoza, J.R., 1997. Acta Cryst. A53, 291-313.) The method, like its parent, is unique that it does not rely on ``back'' transformation from the diffraction pattern into real space and on interpolation within measured data. It is designed to deal successfully with sparse, irregular, incomplete and noisy data. It is also designed to use prior information for ensuring sensible results and for reliable convergence. This article describes the theoretical basis for the reconstruction algorithm, its implementation and quantitative results of tests on synthetic and experimentally obtained data. The program could be used for determining the structure of radiation tolerant samples and, eventually, of large biological molecular structures without the need for crystallization.Comment: 12 pages, 10 figure

    FFAS server: novel features and applications.

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    The Fold and Function Assignment System (FFAS) server [Jaroszewski et al. (2005) FFAS03: a server for profile-profile sequence alignments. Nucleic Acids Research, 33, W284-W288] implements the algorithm for protein profile-profile alignment introduced originally in [Rychlewski et al. (2000) Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Science: a Publication of the Protein Society, 9, 232-241]. Here, we present updates, changes and novel functionality added to the server since 2005 and discuss its new applications. The sequence database used to calculate sequence profiles was enriched by adding sets of publicly available metagenomic sequences. The profile of a user's protein can now be compared with ∼20 additional profile databases, including several complete proteomes, human proteins involved in genetic diseases and a database of microbial virulence factors. A newly developed interface uses a system of tabs, allowing the user to navigate multiple results pages, and also includes novel functionality, such as a dotplot graph viewer, modeling tools, an improved 3D alignment viewer and links to the database of structural similarities. The FFAS server was also optimized for speed: running times were reduced by an order of magnitude. The FFAS server, http://ffas.godziklab.org, has no log-in requirement, albeit there is an option to register and store results in individual, password-protected directories. Source code and Linux executables for the FFAS program are available for download from the FFAS server

    SDSL-ESR-based protein structure characterization

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    As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structur

    Computational Potential Energy Minimization Studies on the Prion AGAAAAGA Amyloid Fibril Molecular Structures

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    X-ray crystallography, NMR (Nuclear Magnetic Resonance) spectroscopy, and dual polarization interferometry, etc are indeed very powerful tools to determine the 3D structures of proteins (including the membrane proteins), though they are time-consuming and costly. However, for some proteins, due to their unstable, noncrystalline and insoluble nature, these tools cannot work. Under this condition, mathematical and physical theoretical methods and computational approaches allow us to obtain a description of the protein 3D structure at a submicroscopic level. This Chapter presents some practical and useful mathematical optimization computational approaches to produce 3D structures of the Prion AGAAAAGA Amyloid Fibrils, from a potential energy minimization point of view. X-ray crystallography finds the X-ray final structure of a protein, which usually need refinements in order to produce a better structure. The computational methods presented in this Chapter can be also acted as a tool for the refinements.Comment: published in [Recent Advances in Crystallography, ISBN: 978-953-51-0754-5, Editor Jason B. Bendict, InTech Open Access Publisher, 19 Sept 2012, hardcover] Chapter 12, DOI: 10.5772/47733, pp.297-312: http://www.intechopen.com/books/recent-advances-in-crystallography/computational-potential-energy-minimization-studies-on-the-prion-agaaaaga-amyloid-fibril-molecular-

    Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA

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    Tuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding
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