Journal of Structural Biology

Finite element modelling of cell wall properties for onion epidermis using a fibre-reinforced hyperelastic model.

Publication Date: 2010 Aug 24 PMID: 20800094
Authors: Qian, M. - Wells, D. M. - Jones, A. - Becker, A.
Journal: J Struct Biol

A combined finite element method and inverse modelling approach is used to model the mechanical deformation of onion epidermis. A fibre-reinforced hyperelastic composite material model considering the fibre distribution has been used to simulate the mechanical behaviour of samples under tension. The mechanical parameters of onion epidermis are determined using an inverse modelling approach. The simulated results show a good correlation with experimental observations.

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Native architecture of the photosynthetic membrane from Rhodobacter veldkampii.

Publication Date: 2010 Aug 23 PMID: 20797440
Authors: Liu, L. N. - Sturgis, J. N. - Scheuring, S.
Journal: J Struct Biol

The photosynthetic membrane in purple bacteria contains several pigment-protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1-RC (light-harvesting complex 1 - reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1-RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb.veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC-LH1-PufX cores, as it has been proposed in Rb. sphaeroides.

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Hysteretic swelling of wood at cellular scale probed by phase contrast X-ray tomography.

Publication Date: 2010 Aug 23 PMID: 20797439
Authors: Derome, D. - Griffa, M. - Koebel, M. - Carmeliet, J.
Journal: J Struct Biol

We investigated the three-dimensional, microscopic, dimensional changes of Picea abies (L. Karst) wood samples due to controlled steps of the ambient relative humidity. The study was performed at the wood cellular scale by high resolution synchroton radiation phase-contrast X-ray tomographic microscopy (srPCXTM). Tomographic images were taken after the samples achieved moisture equilibrium at five adsorption and four desorption steps. For spruce latewood, swelling and shrinkage are found to be larger, more hysteretic and more homomorphic than for earlywood. Furthermore, while latewood undergoes similar strains in the transverse directions, earlywood radial strains are less than a third of the tangential strains. The less homomorphic and smaller swelling/shrinkage of earlywood in radial direction is found to be caused by the presence of rays.

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Releasing of the chromophore from the drug delivery protein C-1027: A molecular dynamics simulations study.

Publication Date: 2010 Aug 21 PMID: 20732428
Authors: Wang, Y. B. - Zhao, X. - Yu, H. - Huang, X. R.
Journal: J Struct Biol

The aromatized chromophore (Chr) of C-1027 with selective DNA-cleaving ability, which is stabilized and delivered by the apoprotein (Apo) in vitro, is not released until the holoprotein (Apo+Chr) penetrates into the cultured cancer cells. As a drug delivery system, the holoprotein has gained much attention in clinical application. However, the Chr-releasing mechanism is ambiguous so far. In this paper, the releasing pathway is investigated using conventional molecular dynamics (MD), essential dynamics (ED), essential dynamics sampling and steered molecular dynamics (SMD) simulations. The results indicate that the releasing paths are related to the local motions of three loops: L3 (Val39-Gln42), L7 (Thr75-Thr79) and L9 (Asn97-Leu100). The major obstacles to Chr releasing come from steric hindrance, direct hydrogen bonds and hydrophobic interactions formed by the three loops, and Ser98 is an important residue in the releasing process. The most favorable direction of releasing is almost parallel to the connection between L7 and L3. Releasing from the direction, Chr only needs to break three hydrogen bonds from Ser98 and Pro76 and the weakest steric hindrance.

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Structure and flexibility in cold-adapted iron superoxide dismutases: The case of the enzyme isolated from Pseudoalteromonas haloplanktis.

Publication Date: 2010 Aug 21 PMID: 20732427
Authors: Merlino, A. - Krauss, I. R. - Castellano, I. - Vendittis, E. D. - Rossi, B. - Conte, M. - Vergara, A. - Sica, F.
Journal: J Struct Biol

Superoxide dismutases (SODs) are metalloenzymes catalysing the dismutation of superoxide anion radicals into molecular oxygen and hydrogen peroxide. Here, we present the crystal structure of a cold-adapted Fe-SOD from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhSOD), and that of its complex with sodium azide. The structures were compared with those of the corresponding homologues having a high sequence identity with PhSOD, such as the mesophilic SOD from Escherichia coli (EcSOD) or Pseudomonas ovalis, and the psychrophilic SOD from Aliivibrio salmonicida (AsSOD). These enzymes shared a large structural similarity, such as a conserved tertiary structure and arrangement of the two monomers, an almost identical total number of inter- and intramolecular hydrogen bonds and salt bridges. However, the two cold-adapted SODs showed an increased flexibility of the active site residues with respect to their mesophilic homologues. Structural information was combined with a characterisation of the chemical and thermal stability performed by CD and fluorescence measurements. Despite of its psychrophilic origin, the denaturation temperature of PhSOD was comparable with that of the mesophilic EcSOD, whereas AsSOD showed a lower denaturation temperature. On the contrary, the values of the denaturant concentration at the transition midpoint were in line with the psychrophilic/mesophilic origin of the proteins. These data provide additional support to the hypothesis that cold-adapted enzymes achieve efficient catalysis at low temperature, by increasing the flexibility of their active site; moreover, our results underline how fine structural modifications can alter enzyme flexibility and/or stability without compromising the overall structure of typical rigid enzymes, such as SODs.

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Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool.

Publication Date: 2010 Aug 21 PMID: 20732426
Authors: Harland, D. P. - Caldwell, J. P. - Woods, J. L. - Walls, R. J. - Bryson, W. G.
Journal: J Struct Biol

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5 degrees . The length of one turn of the helical path of an IF was calculated to be approximately 1mum for an IF tilted at 30 degrees and positioned 100nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.

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Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

Publication Date: 2010 Aug 17 PMID: 20723603
Authors: Nishiyama, H. - Suga, M. - Ogura, T. - Maruyama, Y. - Koizumi, M. - Mio, K. - Kitamura, S. - Sato, C.
Journal: J Struct Biol

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.

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Maximum Likelihood based Classification of Electron Tomographic Data.

Publication Date: 2010 Aug 15 PMID: 20719249
Authors: Stolken, M. - Beck, F. - Haller, T. - Hegerl, R. - Gutsche, I. - Carazo, J. M. - Baumeister, W. - Scheres, S. H. - Nickell, S.
Journal: J Struct Biol

Classification and averaging of sub-tomograms can improve the fidelity and resolution of structures obtained by electron tomography. Here we present a three-dimensional (3D) Maximum Likelihood algorithm - MLTOMO - which is characterized by integrating 3D alignment and classification into a single, unified processing step. The novelty of our approach lies in the way we calculate the probability of observing an individual sub-tomogram for a given reference structure. We assume that the reference structure is affected by a 'compound wedge', resulting from the summation of many individual missing wedges in distinct orientations. The distance metric underlying our probability calculations effectively downweights Fourier components that are observed less frequently. Simulations demonstrate that MLTOMO clearly outperforms the 'constrained correlation' approach and has advantages over existing approaches in cases where the sub-tomograms adopt preferred orientations. Application of our approach to cryo-electron tomographic data of ice-embedded thermosomes revealed distinct conformations that are in good agreement with results obtained by previous single particle studies.

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The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: A bio-polymeric composite.

Publication Date: 2010 Aug 9 PMID: 20705141
Authors: Falini, G. - Sartor, G. - Fabbri, D. - Vergni, P. - Fermani, S. - Belcher, A. M. - Stucky, G. D. - Morse, D. E.
Journal: J Struct Biol

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC-MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.

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Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from Vibrio alginolyticus.

Publication Date: 2010 Aug 10 PMID: 20705140
Authors: Hosogi, N. - Shigematsu, H. - Terashima, H. - Homma, M. - Nagayama, K.
Journal: J Struct Biol

Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32nm.

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Nanoscale morphology of Type I collagen is altered in the Brtl mouse model of Osteogenesis Imperfecta.

Publication Date: 2010 Aug 7 PMID: 20696252
Authors: Wallace, J. M. - Orr, B. G. - Marini, J. C. - Holl, M. M.
Journal: J Struct Biol

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the alpha1 or alpha2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (alpha1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1alpha1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.

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On the evolutionary origins of "Fold Space Continuity": A study of topological convergence and divergence in mixed alpha-beta domains.

Publication Date: 2010 Aug 5 PMID: 20691788
Authors: Sadowski, M. I. - Taylor, W. R.
Journal: J Struct Biol

Existing protein structure classifications group proteins by overall structural similarity at the highest level and by evolutionary relationships at the lowest level, deriving higher-level groups by pairwise structure comparison. For this to be successful requires that large changes in structure are relatively rare in evolution and that proteins with no detectable evolutionary relationship do not converge on similar global chain conformations since this creates conflicts between structural and evolutionary consistency. Analysis of global structural changes using core topological descriptions for 4261 domains from classes C and D of the SCOP database and new measures of topological distance and consistency of classification showed that the topological consistency of SCOP folds is highly variable with some folds having no consistent description and significant overlaps between groups including some members of separate folds with identical topological descriptions. Topological clustering shows that including sufficient indels to allow family members to be joined would also require joining several distinct folds. We conclude that evolutionary changes in the global topology of protein domains are the root cause of many difficulties for present approaches to structure classification using pairwise comparison. As a resolution we propose that a purely structural classification should be created using an approach similar to that adopted by the Gene Ontology in which proteins are assigned labels describing structure.

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New trends in protein expression.

Publication Date: 2010 Aug 4 PMID: 20691273
Authors: Albeck, S. - Nordlund, P. - Sussman, J. L.
Journal: J Struct Biol

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Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa.

Publication Date: 2010 Aug 4 PMID: 20691272
Authors: Alguel, Y. - Lu, D. - Quade, N. - Sauter, S. - Zhang, X.
Journal: J Struct Biol

Pseudomonas aeruginosa is responsible for around 10% of all hospital-acquired infections and the single most important pathogen of cystic fibrosis lungs. P. aeruginosa has high intrinsic and acquired antibiotic resistance, due to the extrusion of antibiotics by multidrug efflux pumps. The gene regulator MexZ controls the expression of mexXY, the efflux pump responsible for resistance to many drugs that are used for treating CF patients. MexZ is shown to be the most frequently mutated gene in P. aeruginosa isolated from CF patient lungs, confirming its importance in multidrug resistance. Here we present the crystal structure of MexZ at 2.9A. Combining the structural information with biochemical data on key mutants identified, we provide an explanation for the structural and functional consequences of these mutants. This work provides a framework for further characterisation of MexZ in order to fully understand its regulation and induction.

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ADP/ATP mitochondrial carrier MD simulations to shed light on the structural-dynamical events that, after an additional mutation, restore the function in a pathological single mutant.

Publication Date: 2010 Aug 3 PMID: 20688168
Authors: Di Marino, D. - Oteri, F. - Morozzo Della Rocca, B. - Chillemi, G. - Falconi, M.
Journal: J Struct Biol

Molecular dynamics simulations of the wild type bovine ADP/ATP mitochondrial carrier, and of the single Ala113Pro and double Ala113Pro/Val180Met mutants, embedded in a lipid bilayer, have been carried out for 30ns to shed light on the structural-dynamical changes induced by the Val180Met mutation restoring the carrier function in the Ala113Pro pathologic mutant. Principal component analysis indicates that, for the three systems, the protein dynamics is mainly characterized by the motion of the matrix loops and of the odd-numbered helices having a conserved proline in their central region. Analysis of the motions shows a different behaviour of single pathological mutant with respect of the other two systems. The single mutation induces a regularization and rigidity of the H3 helix, lost upon the introduction of the second mutation. This is directly correlated to the salt bridge distribution involving residues Arg79, Asp134 and Arg234, hypothesized to interact with the substrate. In fact, in the wild type simulation two stable inter-helices salt bridges, crucial for substrate binding, are present almost over all the simulation time. In line with the impaired ADP transport, one salt interaction is lost in the single mutant trajectory but reappears in the double mutant simulation, where a salt bridge network matching the wild type is restored. Other important structural-dynamical properties, such as the trans-membrane helices mobility, analyzed via the principal component analysis, are similar for the wild type and double mutant while are different for the single mutant, providing a mechanistic explanation for their different functional properties.

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The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.

Publication Date: 2010 Aug 3 PMID: 20688167
Authors: Xiao, R. - Anderson, S. - Aramini, J. - Belote, R. - Buchwald, W. A. - Ciccosanti, C. - Conover, K. - Everett, J. K. - Hamilton, K. - Huang, Y. J. - Janjua, H. - Jiang, M. - Kornhaber, G. J. - Lee, D. Y. - Locke, J. Y. - Ma, L. C. - Maglaqui, M. - Mao, L. - Mitra, S. - Patel, D. - Rossi, P. - Sahdev, S. - Sharma, S. - Shastry, R. - Swapna, G. V. - Tong, S. N. - Wang, D. - Wang, H. - Zhao, L. - Montelione, G. T. - Acton, T. B.
Journal: J Struct Biol

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

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Subtomogram alignment by adaptive Fourier coefficient thresholding.

Publication Date: 2010 Sep PMID: 20621702
Authors: Amat, F. - Comolli, L. R. - Moussavi, F. - Smit, J. - Downing, K. H. - Horowitz, M.
Journal: J Struct Biol

In the past few years, three-dimensional (3D) subtomogram alignment has become an important tool in cryo-electron tomography (CET). This technique allows one to produce higher resolution images of structures which can not be reconstructed using single-particle methods. Building on previous work, we present a new dissimilarity measure between subtomograms that works well for the noisy images that often occur in CET images. A technique that is more robust to noise provides the ability to analyze macromolecules in thicker samples such as whole cells or lower the defocus in thinner samples to push the first zero of the Contrast Transfer Function (CTF). Our method, Threshold Constrained Cross-Correlation (TCCC), uses statistics of the noise to automatically select only a small percentage of the Fourier coefficients to compute the cross-correlation, which has two main advantages: first, it reduces the influence of the noise by looking at only those peaks dominated by signal; and second, it avoids the missing wedge normalization problem since we consider the same number of coefficients for all possible pairs of subtomograms. We present results with synthetic and real data to compare our approach with other existing methods under different SNR and missing wedge conditions, and show that TCCC improves alignment results for datasets with SNR<0.1. We have made our source code freely available for the community.

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Sulfate ion interaction with 'anion recognition' short peptide motif at the N-terminus of an isolated helix: A conformational landscape.

Publication Date: 2010 Sep PMID: 20570734
Authors: Sheet, T. - Banerjee, R.
Journal: J Struct Biol

Anion-binding motifs in proteins are typically conserved in sequence and conformation. Crystal structural studies have shown that such motifs often occur in loop regions preceding a helix and interaction with the anions can induce their well defined conformational changes. In order to understand the properties of such motifs in isolation, we have synthesized an 18-residue chimeric polypeptide whose C-terminal part is a designed helix and its N-terminal consists of a C(alpha)NN anion binding structural motif containing residues Leu-Gly-Lys-Gln (residues 107-110 of protein DNA-glycosylase). We present evidence for the interaction of a sulfate (SO(4)(2-)) ion with the L-G-K-Q segment using complementary spectroscopic techniques. Moreover, upon interaction with SO(4)(2-) ion the N-terminal L-G-K-Q segment undergoes a non-helical to helical transition similar to what is observed in protein crystal structure. This work clearly demonstrates the "local" nature of anion binding and the accompanying conformational change that helps in understanding the influence of sequence/structural context of anion binding in proteins.

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Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.

Publication Date: 2010 Sep PMID: 20570732
Authors: Cogger, V. C. - McNerney, G. P. - Nyunt, T. - DeLeve, L. D. - McCourt, P. - Smedsrod, B. - Le Couteur, D. G. - Huser, T. R.
Journal: J Struct Biol

Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.

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Raman spectroscopic study of the mineral composition of cirratulid tubes (Annelida, Polychaeta).

Publication Date: 2010 Sep PMID: 20566380
Authors: Taylor, P. D. - Vinn, O. - Kudryavtsev, A. - Schopf, J. W.
Journal: J Struct Biol

Raman spectroscopy was used to determine the mineralogical composition of the calcareous tubes of three species belonging to the family Cirratulidae. In all three cases, the tubes were found to be aragonitic, confirming previous inferences based on EDX and thin section studies, and corroborated by new EDX analyses revealing the presence of Sr but no Mg. Biomineralization in cirratulids is first recorded in the Oligocene epoch, at a time of aragonite seas. Similarly, the mineralogies of the earliest skeletons matched seawater chemistry in three other polychaete groups that independently evolved calcareous skeletons.

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4.6A Cryo-EM reconstruction of tobacco mosaic virus from images recorded at 300 keV on a 4k x 4k CCD camera.

Publication Date: 2010 Sep PMID: 20558300
Authors: Clare, D. K. - Orlova, E. V.
Journal: J Struct Biol

Tobacco mosaic virus (TMV) is a plant virus with a highly ordered organisation and has been described in three different structural states: As stacked disks without RNA (X-ray crystallography), as a helical form with RNA (X-ray fibre diffraction) and as a second distinct helical form with RNA (cryo-EM). Here we present a structural analysis of TMV as a test object to assess the quality of cryo-EM images recorded at 300 keV on a CCD camera. The 4.6A TMV structure obtained is consistent with the previous cryo-EM structure and confirms that there is a second helical form of TMV. The structure here also shows that with a similar number of TMV segments an equivalent resolution can be achieved with a 4k CCD camera at 300 keV.

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Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata.

Publication Date: 2010 Sep PMID: 20553887
Authors: Auzoux-Bordenave, S. - Badou, A. - Gaume, B. - Berland, S. - Helleouet, M. N. - Milet, C. - Huchette, S.
Journal: J Struct Biol

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.

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Structure of the novel 14kDa fragment of alpha-subunit of phycoerythrin from the starving cyanobacterium Phormidium tenue.

Publication Date: 2010 Sep PMID: 20546902
Authors: Soni, B. R. - Hasan, M. I. - Parmar, A. - Ethayathulla, A. S. - Kumar, R. P. - Singh, N. K. - Sinha, M. - Kaur, P. - Yadav, S. - Sharma, S. - Madamwar, D. - Singh, T. P.
Journal: J Struct Biol

The rod-like phycobilisome (PBS) in cyanobacterium is the light-harvesting complex of phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). The orderly degradation of PBS was observed under starvation conditions. A 14 kDa truncated fragment of alpha-subunit of PE (F-alphaPE) was identified from the degraded product. F-alphaPE was purified to homogeneity, sequenced and crystallized. The merohedrally twinned crystals with a twinning factor of approximately 0.5 were obtained. The crystal structure of F-alphaPE was determined with molecular replacement method using detwinned data and refined to an R(cryst) factor of 23.2% (R(free)=27.6%). The structure consisted of two crystallographically independent molecules in the asymmetric unit. The two molecules were designated as molecules A and B with a buried area of 200 A(2) at the interface. The structure of F-alphaPE consists of seven alpha-helices A, B, E, F, F', G and H. The first 31N-terminal residues that fold into parallel alpha-helices X and Y in other PEs are not present in the amino acid sequence of F-alphaPE. Both molecules, A and B contain two chromophore ligands, PEB1 and PEB2 in each. These are covalently linked to the polypeptide chain through Cys82 and Cys139, respectively. The superimposition of C(alpha) tracings of molecules A and B shows an r.m.s. shift of 1.0 A indicating that the structures of two independent molecules are very similar. The degradation of phycobilisome proteins under starvation stress seems to occur to supplement the requirement of amino acids for protein synthesis and to reduce the absorption of light energy.

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Crystal structures of a novel anti-HIV mannose-binding lectin from Polygonatum cyrtonema Hua with unique ligand-binding property and super-structure.

Publication Date: 2010 Sep PMID: 20546901
Authors: Ding, J. - Bao, J. - Zhu, D. - Zhang, Y. - Wang, D. C.
Journal: J Struct Biol

Polygonatum cyrtonema lectin (PCL) is a novel anti-HIV mannose-binding lectin from Galanthus nivalis agglutinin (GNA)-related lectin family. Crystal structures of ligand-free PCL and its complexes with monomannoside and alpha1-3 dimannoside have been determined. The ligand-free PCL is dimeric, with both subunits adopt the beta-prism II fold. PCL subunit binds mannose using a potential bivalent mode instead of the usual trivalent mode, in which carbohydrate-binding site (CBS) I and CBS III adopt the conserved mannose-binding motif of QXDXNXVXY (X is one of any amino acid residues) as observed in other structurally characterized GNA-related lectins, while CBS II adopts a modified motif with residues Gln58 and Asp60, which are critical for mannose-binding, substituted by His58 and Asn60, respectively. As a result, CBS II is unfit for mannose-binding. In the mannoside complexes, ligand-bindings only occur at CBS I which provides the specificity for alpha1-3 dimannoside. CBS II and CBS III are cooperatively occupied by a well-ordered sulfate ion, through which the individual dimers are cross-linked to form a unique super-structure of 3(2) helical lattice. Surveying the sequences of GNA-related lectins revealed that the modified binding motif of CBS II is widely distributed in the Liliaceae family as an intrinsic structural element. There is evidence that other GNA-related lectins will also adopt the similar super-structure as PCL. Thus PCL structure, unique in ligand-binding mode, may represent a novel type of structure of GNA-related lectins. Comparative analyses indicated that the dimer-based super-structure may play a primary role in the anti-HIV property of PCL.

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X-ray microCT imaging technique reveals corm microstructures of an arctic-boreal cotton-sedge, Eriophorum vaginatum.

Publication Date: 2010 Sep PMID: 20541609
Authors: Bogart, S. J. - Spiers, G. - Cholewa, E.
Journal: J Struct Biol

X-ray computed tomography (CT), a non-destructive imaging technique, has recently been effectively applied to botanical research. In this study an X-ray microCT technique was developed to allow for anatomical study of the overwintering corms of Eriophorum vaginatum, an ecologically important sedge species in arctic tussock-tundra and boreal peatlands. Using a GE Medical MS8X-130 X-ray microCT scanner, optimal imaging parameters included scanning isolated corms at 80 k Vp and 100 microA with a 3500 ms exposure time and an isotropic voxel size of 10 microm. A Gaussian blur image filter with a blur radius (sigma) of two pixels was applied to the optimal dataset to improve visual detection and contrast of tissues while removing 99.2% of image noise. Using the developed X-ray microCT technique several undocumented anatomical characteristics of the corm were identified including the vascular connection between a parent corm and branching cormel and the 3D shape of sclereid clusters. The 3D structure of sclereid clusters was determined whereby the perimeter of their lance shape is greatly reinforced by sclereids with thicker secondary cell walls as compared to those of the interior of the cluster. The structure of sclereid clusters and their association with leaf traces suggests they may be stabilizing the corm-leaf connection to protect vascular tissues from physical damage. The proposed X-ray microCT technique is an excellent tool for determination of the 3D structure of E. vaginatum corms and may be used to detect alterations in tissue structure and chemistry in response to environmental change in this and other Cyperaceous species.

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Cryo-electron tomography of bacteriophage phi6 procapsids shows random occupancy of the binding sites for RNA polymerase and packaging NTPase.

Publication Date: 2010 Sep PMID: 20538059
Authors: Nemecek, D. - Heymann, J. B. - Qiao, J. - Mindich, L. - Steven, A. C.
Journal: J Struct Biol

Assembly of dsRNA bacteriophage phi6 involves packaging of the three mRNA strands of the segmented genome into the procapsid, an icosahedrally symmetric particle with recessed vertices. The hexameric packaging NTPase (P4) overlies these vertices, and the monomeric RNA-dependent RNA polymerase (RdRP, P2) binds at sites inside the shell. P2 and P4 are present in substoichiometric amounts, raising the questions of whether they are recruited to the nascent procapsid in defined amounts and at specific locations, and whether they may co-localize to form RNA-processing assembly lines at one or more "special" vertices. We have used cryo-electron tomography to map both molecules on individual procapsids. The results show variable complements that accord with binomial distributions with means of 8 (P2) and 5 (P4), suggesting that they are randomly incorporated in copy numbers that simply reflect availability, i.e. their rates of synthesis. Analysis of the occupancy of potential binding sites (20 for P2; 12 for P4) shows no tendency to cluster nor for P2 and P4 to co-localize, suggesting that the binding sites for both proteins are occupied in random fashion. These observations indicate that although P2 and P4 act sequentially on the same substrates there is no direct physical coupling between their activities.

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An adaptive Expectation-Maximization algorithm with GPU implementation for electron cryomicroscopy.

Publication Date: 2010 Sep PMID: 20538058
Authors: Tagare, H. D. - Barthel, A. - Sigworth, F. J.
Journal: J Struct Biol

Maximum-likelihood (ML) estimation has very desirable properties for reconstructing 3D volumes from noisy cryo-EM images of single macromolecular particles. Current implementations of ML estimation make use of the Expectation-Maximization (EM) algorithm or its variants. However, the EM algorithm is notoriously computation-intensive, as it involves integrals over all orientations and positions for each particle image. We present a strategy to speedup the EM algorithm using domain reduction. Domain reduction uses a coarse grid to evaluate regions in the integration domain that contribute most to the integral. The integral is evaluated with a fine grid in these regions. In the simulations reported in this paper, domain reduction gives speedups which exceed a factor of 10 in early iterations and which exceed a factor of 60 in terminal iterations.

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Crystal structure of a fructokinase homolog from Halothermothrix orenii.

Publication Date: 2010 Sep PMID: 20493950
Authors: Chua, T. K. - Seetharaman, J. - Kasprzak, J. M. - Ng, C. - Patel, B. K. - Love, C. - Bujnicki, J. M. - Sivaraman, J.
Journal: J Struct Biol

Fructokinase (FRK; EC 2.7.1.4) catalyzes the phosphorylation of d-fructose to d-fructose 6-phosphate (F6P). This irreversible and near rate-limiting step is a central and regulatory process in plants and bacteria, which channels fructose into a metabolically active state for glycolysis. Towards understanding the mechanism of FRK, here we report the crystal structure of a FRK homolog from a thermohalophilic bacterium Halothermothrixorenii (Hore_18220 in sequence databases). The structure of the Hore_18220 protein reveals a catalytic domain with a Rossmann-like fold and a beta-sheet "lid" for dimerization. Based on comparison of Hore_18220 to structures of related proteins, we propose its mechanism of action, in which the lid serves to regulate access to the substrate binding sites. Close relationship of Hore_18220 and plant FRK enzymes allows us to propose a model for the structure and function of FRKs.

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Visualization of inositol 1,4,5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy.

Publication Date: 2010 Sep PMID: 20457258
Authors: Cardenas, C. - Escobar, M. - Garcia, A. - Osorio-Reich, M. - Hartel, S. - Foskett, J. K. - Franzini-Armstrong, C.
Journal: J Struct Biol

The receptors for the second messenger InsP(3) comprise a family of closely related ion channels that release Ca(2+) from intracellular stores, most prominently the endoplasmic reticulum and its extension into the nuclear envelope. The precise sub-cellular localization of InsP(3)Rs and the spatial relationships among them are important for the initiation, spatial and temporal properties and propagation of local and global Ca(2+) signals, but the spatial organization of InsP(3)Rs in Ca(2+) stores is poorly characterized. Using nuclei isolated from insect Sf9 cells and freeze-dry rotary shadowing, we have addressed this by directly visualizing the cytoplasmic domain of InsP(3)R located on the cytoplasmic side of the nuclear envelope. Identification of approximately 15 nm structures as the cytoplasmic domain of InsP(3)R was indirectly supported by a marked increase in their frequency after transient transfections with cDNAs for rat types 1 and 3 InsP(3)R, and directly confirmed by gold labeling either with heparin or a specific anti-InsP(3)R antibody. Over-expression of InsP(3)R did not result in the formation of arrays or clusters with channels touching each other. Gold-labeling suggests that the channel amino terminus resides near the center of the cytoplasmic tetrameric quaternary structure. The combination of nuclear isolation with freeze-drying and rotary shadow techniques allows direct visualization of InsP(3)Rs in native nuclear envelopes and can be used to determine their spatial distribution and density.

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Structural determinants stabilizing helical distortions related to proline.

Publication Date: 2010 Sep PMID: 20457257
Authors: Rey, J. - Deville, J. - Chabbert, M.
Journal: J Struct Biol

The distortions related to proline in a alpha-helix can be accommodated by different structural elements. To obtain an exhaustive view of these distortions, we data mined a non-redundant subset of the Protein Data Bank in search of proline residues included either within contiguous helices or within structural motifs in which two helices are joined by a few linker residues with backbone dihedral angles in the generous alpha region. The distortions correspond to "typical" and "non-typical" proline distortions, with relative ratio of 0.65 and 0.35, respectively. Analysis of "non-typical" proline distortions indicates that most linkers have one (75%) or two residues (20%) and that proline is preferentially located at the second or third position of the second helix (95%). The dihedral angles of the linker residues are located in two areas of the generous alpha region. Structures with linker(s) in the alpha1 area, which is characterized by very negative phi values, possess i to i-5 H-bonds and correspond to pi bulges. Structures with linker(s) in the alpha2 area, which links the alpha and beta regions, possess i to i-3 H-bonds and correspond to tight turns. Further classification of bulges and turns as a function of the linker length and proline position yields five canonical structures, representing 85% of "non-typical" proline distortions. These structures are characterized by distinct H-bonding patterns and structural determinants and correspond to different classes of pi bulges and tight turns. This hierarchical approach provides a straightforward and robust classification of proline-related helical distortions.

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Initial stages of enamel erosion: An in situ atomic force microscopy study.

Publication Date: 2010 Sep PMID: 20438849
Authors: Parkinson, C. R. - Shahzad, A. - Rees, G. D.
Journal: J Struct Biol

Time-resolved in situ atomic force microscopy has been employed to examine erosion progression in untreated and fluoride-treated enamel specimens during exposure to citric acid. Contact with the acidic reaction solution initiated the emergence and growth of dissolution pits in both the native and the fluoride-treated enamel. In native enamel, pits are first observed after 90 min exposure to the reaction solution, compared to 250 min in the case of the fluoride-treated enamel. These findings indicate that, within the constraints of this study, a single application of fluoride solution (1000 mg/L, 2 min) confers protection to the enamel surface against acid-mediated erosion. This paper also highlights the potential role intrinsic defects may have on the susceptibility of enamel to erosion and, in part, may explain why some people are more susceptible to acid erosion.

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Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1.

Publication Date: 2010 Sep PMID: 20438847
Authors: Li, H. - Shi, H. - Wang, H. - Zhu, Z. - Li, X. - Gao, Y. - Cui, Y. - Niu, L. - Teng, M.
Journal: J Struct Biol

Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein that contains three RNA recognition motif (RRM) domains (termed Pub1RRM1, Pub1RRM2 and Pub1RRM3). Pub1 has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNA decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. RNA-binding analyses showed that Pub1 binds to poly(U) in vitro. Here we show the crystal structures of Pub1RRM2 and the first two tandem RRM domains (Pub1RRM12). Crystallography showed that the structure of Pub1RRM12 is a domain-swapped dimer. Size exclusion chromatography assay and analytical ultracentrifugation (AUC) showed that Pub1RRM12 is a monomer in solution. Kinetic analysis showed that all three individual RRM domains can bind to poly(U) with similar affinities and Pub1RRM12 binds to a long poly(U) segment with higher affinity. Mutagenesis analysis revealed that residues on the beta-sheets of Pub1RRM1 and Pub1RRM2 are critical for poly(U) binding.

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Quantitative microstructural studies of the armor of the marine threespine stickleback (Gasterosteus aculeatus).

Publication Date: 2010 Sep PMID: 20433929
Authors: Song, J. - Reichert, S. - Kallai, I. - Gazit, D. - Wund, M. - Boyce, M. C. - Ortiz, C.
Journal: J Struct Biol

In this study, a quantitative investigation of the microstructure and composition of field-caught marine Gasterosteus aculeatus (threespine stickleback) armor is presented, which provides useful phylogenetic information and insights into biomechanical function. Micro-computed tomography (microCT) was employed to create full three-dimensional images of the dorsal spines and basal plate, lateral plates, pelvic girdle and spines and to assess structural and compositional properties such as the spatial distribution of thickness (approximately 100-300 microm), the heterogeneous cross-sectional geometry (centrally thickened), plate-to-plate juncture and overlap (approximately 50% of the plate width), and bone mineral density (634-748 HA/cm(3)). The convolution of plate geometry in conjunction with plate-to-plate overlap allows a relatively constant armor thickness to be maintained throughout the assembly, promoting spatially homogeneous protection and thereby avoiding weakness at the armor unit interconnections. Plate-to-plate junctures act to register and join the plates while permitting compliance in sliding and rotation in selected directions. Mercury porosimetry was used to determine the pore size distribution and volume percent porosity of the lateral plates (20-35 vol.%) and spines (10-15 vol.%). SEM and microCT revealed a porous, sandwich-like cross-section beneficial for bending stiffness and strength at minimum weight. Back-scattered electron microscopy and energy dispersive X-ray analysis were utilized to quantify the weight percent mineral content (58-68%). Scanning electron microscopy and surface profilometry were used to characterize the interior and exterior surface topography (tubercles) of the lateral plates. The results obtained in this study are discussed in the context of mechanical function, performance, fitness, and survivability.

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Crystallographic analysis reveals a unique lidocaine binding site on human serum albumin.

Publication Date: 2010 Sep PMID: 20347991
Authors: Hein, K. L. - Kragh-Hansen, U. - Morth, J. P. - Jeppesen, M. D. - Otzen, D. - Moller, J. V. - Nissen, P.
Journal: J Struct Biol

Human serum albumin (HSA), the major protein component in blood plasma and in extravascular spaces, is known to participate in the binding and transport of a variety of endogenous and exogenous organic compounds with anionic or electronegative features. We here report on the 3.3A resolution crystal structure of HSA complexed with the cationic, and widely used, anesthetic lidocaine. We find that lidocaine and HSA co-crystallise as a dimer in the unusual space group I4(1). The dimer consists of one HSA molecule without ligand and one HSA molecule with a single, bound lidocaine. HSA is a heart-shaped protein composed of three homologous helical domains (I-III), which can be subdivided into two subdomains (A and B), and lidocaine binds to a unique site formed by residues from subdomain IB facing the central, interdomain crevice. In the crystal, binding seems to introduce only local conformational changes in the protein. According to intrinsic fluorescence experiments with aqueous HSA binding results in widespread conformational changes involving Trp214 in subdomain IIA. Results obtained with equilibrium dialysis and isothermal titration calorimetry show that lidocaine binding is of a low affinity and occurs at one discrete binding site in accordance with the X-ray data. Another crystal form of ligand-free HSA obtained in the presence of ammonium sulphate was determined at 2.3A resolution revealing a sulphate ion accepting cavity at the surface of subdomain IIIA. The present results contribute to a further characterisation of the exceptional binding properties of HSA.

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