The Experts below are selected from a list of 8127 Experts worldwide ranked by ideXlab platform
John L Rubinstein - One of the best experts on this subject based on the ideXlab platform.
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structure of intact thermus thermophilus v atpase by cryo em reveals organization of the membrane bound vo motor
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: John L RubinsteinAbstract:Abstract The eubacterium Thermus thermophilus uses a Macromolecular Assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 A resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the Assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the VO region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in VO gives insight into the mechanism of torque generation from proton translocation.
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structure of intact thermus thermophilus v atpase by cryo em reveals organization of the membrane bound v o motor
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Wilson C Y Lau, John L RubinsteinAbstract:The eubacterium Thermus thermophilus uses a Macromolecular Assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at approximately 16 A resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the Assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V(O) region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V(O) gives insight into the mechanism of torque generation from proton translocation.
Frank Dimaio - One of the best experts on this subject based on the ideXlab platform.
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automated structure refinement of Macromolecular assemblies from cryo em maps using rosetta
eLife, 2016Co-Authors: Ray Yuruei Wang, Yifan Song, Benjamin A Barad, Yifan Cheng, J S Fraser, Frank DimaioAbstract:Cryo-EM has revealed the structures of many challenging yet exciting Macromolecular assemblies at near-atomic resolution (3-4.5A), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids - typical in a Macromolecular Assembly - is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.
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automated structure refinement of Macromolecular assemblies from cryo em maps using rosetta
bioRxiv, 2016Co-Authors: Ray Yuruei Wang, Yifan Song, Benjamin A Barad, Yifan Cheng, J S Fraser, Frank DimaioAbstract:Cryo-EM has revealed many challenging yet exciting Macromolecular assemblies at near-atomic resolution (3-4.5 Angstrom), providing biological phenomena with molecular descriptions. However, at these resolutions accurately positioning individual atoms remains challenging and may be error-prone. Manually refining thousands of amino acids -- typical in a Macromolecular Assembly -- is tedious and time-consuming. We present an automated method that can improve the atomic details in models manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining or improving the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.
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a refined model of the prototypical salmonella spi 1 t3ss basal body reveals the molecular basis for its Assembly
PLOS Pathogens, 2013Co-Authors: Julien R C Bergeron, L J Worrall, Nikolaos G Sgourakis, Frank Dimaio, Richard A Pfuetzner, Heather B Felise, M Vuckovic, Angel C Yu, Samuel I Miller, David BakerAbstract:The T3SS injectisome is a syringe-shaped Macromolecular Assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric Assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body Assembly, and notably demonstrate a conserved oligomerization mechanism.
Brett M Collins - One of the best experts on this subject based on the ideXlab platform.
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applications of isothermal titration calorimetry in pure and applied research survey of the literature from 2010
Journal of Molecular Recognition, 2012Co-Authors: Rajesh Ghai, Robert J Falconer, Brett M CollinsAbstract:Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for Macromolecular Assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein a-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method. Copyright (C) 2011 John Wiley & Sons, Ltd.
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applications of isothermal titration calorimetry in pure and applied research survey of the literature from 2010
Journal of Molecular Recognition, 2012Co-Authors: Rajesh Ghai, Robert J Falconer, Brett M CollinsAbstract:Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for Macromolecular Assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.
F A Goldbaum - One of the best experts on this subject based on the ideXlab platform.
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high order quaternary arrangement confers increased structural stability to brucella sp lumazine synthase
Journal of Biological Chemistry, 2004Co-Authors: Vanesa Zylberman, Ana Cauerhff, Bradford C Braden, Patricio O Craig, Sebastian Klinke, F A GoldbaumAbstract:The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary Assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this Macromolecular Assembly. The higher molecular order of BLS increases its stability 20 °C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins.
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divergence in Macromolecular Assembly x ray crystallographic structure analysis of lumazine synthase from brucella abortus
Journal of Molecular Biology, 2000Co-Authors: Bradford C Braden, Igor Polikarpov, Ana Cauerhff, C A Velikovsky, F A GoldbaumAbstract:We have determined the three-dimensional structure of 6, 7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals. This enzyme catalyses the formation of 6, 7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. The three-dimensional X-ray crystal structure of the enzyme from B. abortus has been solved and refined at 2.7 A resolution to a final R-value of 0.18 (R(free)=0.23). The Macromolecular Assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known. While the protein from B. subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B. abortus is pentameric in its crystalline form. Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range. Furthermore, we compare the structures of the enzyme from B. subtilis and B. abortus and describe the C teminus structure which accounts for the differences in quaternary structure.
Maneesha S Inamdar - One of the best experts on this subject based on the ideXlab platform.
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networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in drosophila nephrocytes
Experimental Cell Research, 2018Co-Authors: Simi Muraleedharan, Aksah Sam, Helen Skaer, Maneesha S InamdarAbstract:Abstract Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional Macromolecular Assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization and endoplasmic reticulum structure, are required to position the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.
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networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in drosophila nephrocytes
bioRxiv, 2016Co-Authors: Simi Muraleedharan, Helen Skaer, Maneesha S InamdarAbstract:Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional Macromolecular Assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the nephrocyte architecture and elements that maintain the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization is required for positioning the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.
