Journal Articles

  • Stretching desmin filaments with receding meniscus reveals large axial tensile strength
    [Apr 2014]

    Publication date: Available online 16 April 2014
    Source:Journal of Structural Biology

    Author(s): Balázs Kiss , Miklós S.Z. Kellermayer

    Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin’s elasticity is not fully understood. We explored desmin elasticity by molecular combing filaments with forces calculated to be as large as 4nN. Average filament contour length increased 1.55-fold axial on average. Molecular combing together with EGTA-treatment caused the fragmentation of the filament into short, 60 to 120-nm-long and 4-nm-wide structures. The fragments display a surface periodicity of 38nm, suggesting that they are composed of laterally attached desmin dimers. The axis of the fragments may deviate significantly from that of the overstretched filament, indicating that they have a large orientational freedom in spite of being axially interconnected. The emergence of protofibril fragments thus suggests that the interconnecting head or tail domains of coiled-coil desmin dimers are load-bearing elements during axial stretch.





    Categories: Journal Articles
  • Structural insights into regulatory mechanisms of MO25-mediated kinase activation
    [Apr 2014]

    Publication date: Available online 16 April 2014
    Source:Journal of Structural Biology

    Author(s): Qian Hao , Miao Feng , Zhubing Shi , Chuanchuan Li , Min Chen , Wenjia Wang , Meng Zhang , Shi Jiao , Zhaocai Zhou

    The tumor suppressor kinase LKB1 and germinal center kinases (GCKs) are key regulators of various cellular functions. The adaptor molecule MO25 not only recruits and activates LKB1 through the pseudokinase STRAD, but also may directly activate GCKs like MST3, MST4, STK25, OSR1 and SPAK. Targeting MO25 in a pathological setting has been recently studied in mouse. Yet the regulatory mechanism of MO25-mediated kinase activation is not fully understood. Here, our structural studies of MO25-related kinases reveal that MO25 binds to and activates GCK kinases or pseudokinase through a unified structural mechanism, featuring an active conformation of the αC helix and A-loop stabilized by MO25. Compared to GCKs that are directly activated by MO25-binding, activation of LKB1 has evolved additional layer of regulatory machinery, i.e., MO25 “activates” the pseudokinase STRAD, which in turn activates LKB1. Importantly, the structures of MO25–STK25 and MO25–MST3 determined in this work represent a transition/intermediate state and a fully activated state, respectively during the MO25-mediated kinase activating process.





    Categories: Journal Articles
  • Nano-thrombelastography of fibrin during blood plasma clotting
    [Apr 2014]

    Publication date: Available online 13 April 2014
    Source:Journal of Structural Biology

    Author(s): Tímea Feller , Miklós S.Z. Kellermayer , Balázs Kiss

    Hemostasis is a complex process that relies on the sensitive balance between the formation and breakdown of the thrombus, a three-dimensional polymer network of the fibrous protein fibrin. Neither the details of the fibrinogen–fibrin transition, nor the exact mechanisms of fibrin degradation are fully understood at the molecular level. In the present work we investigated the nanoscale-changes in the viscoelasticity of the 3D-fibrin network during fibrinogenesis and streptokinase (STK)-induced fibrinolysis by using a novel application of force spectroscopy, named nano-thrombelastography. In this method the changes in the bending of an oscillating atomic-force-microscope (AFM) cantilever in human blood–plasma droplet were followed as a function of time. Whereas the global features of the time-dependent change in cantilever deflection corresponded well to a macroscopic thrombelastogram, the underlying force spectra revealed large, sample-dependent oscillations in the range of 3–50nN and allowed the separation of elastic and viscous components of fibrin behavior. Upon STK treatment the nano-thrombelastogram signal decayed gradually. The decay was driven by a decrease in thrombus elasticity, whereas thrombus viscosity decayed with a time delay. In scanning AFM images mature fibrin appeared as 17-nm-high and 12–196-nm-wide filaments. STK-treatment resulted in the decrease of filament height and the appearance of a surface roughness with 23.7nm discrete steps that corresponds well to the length of a fibrinogen monomer. Thus, the initial decay of thrombus elasticity during fibrinolysis may be caused by the axial rupture of fibrin fibers.





    Categories: Journal Articles
  • A method for 3D-reconstruction of a muscle thick filament using the tilt series images of a single filament electron tomogram
    [Apr 2014]

    Publication date: Available online 12 April 2014
    Source:Journal of Structural Biology

    Author(s): G. Márquez , A. Pinto , L. Alamo , B. Baumann , F. Ye , H. Winkler , K. Taylor , R. Padrón

    Myosin interacting-heads (MIH) motifs are visualized in 3D-reconstructions of thick filaments from striated muscle. These reconstructions are calculated by averaging methods using images from electron micrographs of grids prepared using numerous filament preparations. Here we propose an alternative method to calculate the 3D-reconstruction of a single thick filament using only a tilt series images recorded by electron tomography. Relaxed thick filaments, prepared from tarantula leg muscle homogenates, were negatively stained. Single-axis tilt series of single isolated thick filaments were obtained with the electron microscope at a low electron dose, and recorded on a CCD camera by electron tomography. An IHRSR 3D-recontruction was calculated from the tilt series images of a single thick filament. The reconstruction was enhanced by including in the search stage dual tilt image segments while only single tilt along the filament axis is usually used, as well as applying a band pass filter just before the back projection. The reconstruction from a single filament has a 40Å resolution and clearly shows the presence of MIH motifs. In contrast, the electron tomogram 3D-reconstruction of the same thick filament – calculated without any image averaging and/or imposition of helical symmetry – only reveals MIH motifs infrequently. This is – to our knowledge – the first application of the IHRSR method to calculate a 3D reconstruction from tilt series images. This single filament IHRSR reconstruction method (SF-IHRSR) should provide a new tool to assess structural differences between well-ordered thick (or thin) filaments in a grid by recording separately their electron tomograms.





    Categories: Journal Articles
  • Identification of the chemical form of sulfur compounds in the Japanese pink coral (Corallium elatius) skeleton using μ-XRF/XAS speciation mapping
    [Apr 2014]

    Publication date: Available online 12 April 2014
    Source:Journal of Structural Biology

    Author(s): Yusuke Tamenori , Toshihiro Yoshimura , Nguyen Trong Luan , Hiroshi Hasegawa , Atsushi Suzuki , Hodaka Kawahata , Nozomu Iwasaki

    The distributions and chemical forms of sulfur compounds in the skeleton of Japanese pink coral (Corallium elatius) were investigated using X-ray spectroscopic techniques combined with micro-focused soft X-ray radiation. Microscopic X-ray fluorescence/soft X-ray photoabsorption (μ-XRF/XAS) speciation mapping clarified that sulfate is the primary species in the coral skeleton, with minor amounts of organic sulfur, whereas both sulfate and organic sulfur coexist in coenenchyme. Analysis of the post-edge region of the XAS spectra confirmed that sulfate ions in the coral skeleton are mainly in the form of gypsum-like inorganic sulfate substituting for the carbonate ions in the calcite skeleton. The sulfate concentration was negatively correlated with the magnesium concentration and positively correlated with that of phosphorus. Speciation mapping of sulfate in the coral skeleton showed clear fluctuations with sulfate concentrations being higher at dark bands, whereas the small amount of organic sulfur had unclear dark/bright bands. These results suggest that the little organic sulfur that is present is contained in the organic matter embedded in the biocrystal of coral skeleton.





    Categories: Journal Articles
  • Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: Towards their structure stability and homogeneity
    [Apr 2014]

    Publication date: Available online 5 April 2014
    Source:Journal of Structural Biology

    Author(s): Susana Galante-Oliveira , Raquel Marçal , Fernanda Guimarães , Jorge Soares , José Carlos Lopes , Jorge Machado , Carlos Barroso

    Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures’ mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates – aragonite and calcite – coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000cm−1 consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.





    Categories: Journal Articles
  • A first census of collagen interruptions: Collagen’s own stutters and stammers
    [Apr 2014]

    Publication date: Available online 4 April 2014
    Source:Journal of Structural Biology

    Author(s): Jordi Bella

    The repetitive Gly-X-Y sequence is the telltale sign of triple helical domains in collagens and collagen-like proteins. Most collagen sequences contain sporadic interruptions of this pattern, which may have functional roles in molecular flexibility, assembly or molecular recognition. However, the structural signatures of the different interruptions are not well defined. Here, a first comprehensive survey of collagen interruptions on collagen sequences from different taxonomic groups is presented. Amino acid preferences at the sites of interruption and the flanking triplets are analysed separately for metazoan and prokaryotic collagens and the concept of commensurateness between interruptions is introduced. Known structural information from model peptides is used to present a common framework for hydrogen bonding topology and variations in superhelical twist for the different types of interruptions. Several collagen interruptions are further classified here as stutters or stammers in analogy to the heptad breaks observed in alpha-helical coiled coils, and the structural consequences of commensurate interruptions in heterotrimeric collagens are briefly discussed. Data presented here will be useful for further investigation on the relation between structure and function of collagen interruptions.





    Categories: Journal Articles
  • Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography
    [Apr 2014]

    Publication date: Available online 2 April 2014
    Source:Journal of Structural Biology

    Author(s): Erik Bos , Leonie Hussaarts , Jan R.T. van Weering , Mark H. Ellisman , Heidi de Wit , Abraham J. Koster

    We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (vitrification of sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.





    Categories: Journal Articles
  • The structure of AAVrh32.33, a novel gene delivery vector
    [Apr 2014]

    Publication date: Available online 2 April 2014
    Source:Journal of Structural Biology

    Author(s): Kyle Mikals , Hyun-Joo Nam , Kim Van Vliet , Luk H. Vandenberghe , Lauren E. Mays , Robert McKenna , James M. Wilson , Mavis Agbandje-McKenna

    The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40–70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1









    Categories: Journal Articles
  • Table of Contents / barcode
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1









    Categories: Journal Articles
  • Automated particle picking for low-contrast macromolecules in cryo-electron microscopy
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Robert Langlois , Jesper Pallesen , Jordan T. Ash , Danny Nam Ho , John L. Rubinstein , Joachim Frank

    Cryo-electron microscopy is an increasingly popular tool for studying the structure and dynamics of biological macromolecules at high resolution. A crucial step in automating single-particle reconstruction of a biological sample is the selection of particle images from a micrograph. We present a novel algorithm for selecting particle images in low-contrast conditions; it proves more effective than the human eye on close-to-focus micrographs, yielding improved or comparable resolution in reconstructions of two macromolecular complexes.





    Categories: Journal Articles
  • Single particle analysis integrated with microscopy: A high-throughput approach for reconstructing icosahedral particles
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Xiaodong Yan , Giovanni Cardone , Xing Zhang , Z. Hong Zhou , Timothy S. Baker

    In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.





    Categories: Journal Articles
  • Key structural arrangements at the C-terminus domain of CETP suggest a potential mechanism for lipid-transfer activity
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Victor García-González , Nadia Gutiérrez-Quintanar , Paola Mendoza-Espinosa , Pilar Brocos , Ángel Piñeiro , Jaime Mas-Oliva

    The cholesteryl-ester transfer protein (CETP) promotes cholesteryl-ester and triglyceride transfer between lipoproteins. We evaluated the secondary structure stability of a series of small peptides derived from the C-terminus of CETP in a wide range of pH’s and lipid mixtures, and studied their capability to carry out disorder-to-order secondary structure transitions dependent of lipids. We report that while a mixture of phosphatidylcholine/cholesteryl-esters forms large aggregated particles, the inclusion of a series of CETP carboxy-terminal peptides in a stable α-helix conformation, allows the formation of small homogeneous micelle-like structures. This phenomenon of lipid ordering was directly connected to secondary structural transitions at the C-terminus domain when lysophosphatidic acid and lysophosphatidylcholine lipids were employed. Circular dichroism, cosedimentation experiments, electron microscopy, as well as molecular dynamics simulations confirm this phenomenon. When purified CETP is studied, the same type of phenomenon occurs by promoting the reorganization of lipid from large to smaller particles. Our findings extend the emerging view for a novel mechanism of lipid transfer carried out by CETP, assigning its C-terminus domain the property to accomplish lipid ordering through secondary structure disorder-to-order transitions.





    Categories: Journal Articles
  • Local regularization of tilt projections reduces artifacts in electron tomography
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Mauro Maiorca , Coralie Millet , Eric Hanssen , Brian Abbey , Edmund Kazmierczak , Leann Tilley

    Electron tomography produces very high resolution 3D image volumes useful for investigating the structure and function of cellular components. Unfortunately, unavoidable discontinuities and physical constraints in the acquisition geometry lead to a range of artifacts that can affect the reconstructed image. In particular, highly electron dense regions, such as gold nanoparticles, can hide proximal biological structures and degrade the overall quality of the reconstructed tomograms. In this work we introduce a pre-reconstruction non-conservative non-linear isotropic diffusion (NID) filter that automatically identifies and reduces local irregularities in the tilt projections. We illustrate the improvement in quality obtained using this approach for reconstructed tomograms generated from samples of malaria parasite-infected red blood cells. A quantitative and qualitative evaluation for our approach on both simulated and real data is provided.





    Categories: Journal Articles
  • Structural and functional characterization of enamel pigmentation in shrews
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): M. Dumont , T. Tütken , A. Kostka , M.J. Duarte , S. Borodin

    Pigmented tooth enamel occurs in several vertebrate clades, ranging from mammals to fish. Although an iron compound is associated with this orange to red colored pigmentation, its chemical and structural organization within the enamel is unknown. To determine the nature of the iron compound, we investigated heavily pigmented teeth of the northern short-tailed shrew Blarina brevicauda using combined characterization techniques such as scanning and transmission electron microscopy and synchrotron X-ray diffraction. We found that the pigmentation of the enamel with an iron content of around 8wt% results from a close to amorphous magnetite phase deposited around the nm-sized enamel crystals. Furthermore, the influence of the pigmentation on the enamel hardness was determined by nanoindentation measurements. Finally, the biomechanical function and biological context are discussed in light of the obtained results.





    Categories: Journal Articles
  • Robust membrane detection based on tensor voting for electron tomography
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Antonio Martinez-Sanchez , Inmaculada Garcia , Shoh Asano , Vladan Lucic , Jose-Jesus Fernandez

    Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure.





    Categories: Journal Articles
  • Motif Analyzer for protein 3D structures
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Evgeniy Aksianov

    The topology of the protein structure of all-β- or α/β-class is a special arrangement of β-strands within β-sheets (and α-helices surrounding β-sheets) and the order of them along the polypeptide chain. Structural motifs are a subset of strands and/or helices with widely spread topology. Structural motifs are used for classification of protein structure. Because of an increasing variety of known structures, an automatic tool for motif detection is needed. MotAn is an algorithmic detector of structural motifs in a given 3D protein structure. It detects β-hairpins, β-meanders, β-helices, Greek keys, interlocks, jellyrolls, β-α-β-motifs and β-α-β-helices. MotAn was tested on selected SCOP families and shown to be more sensitive detector than the PTGL and PROMOTIF programs. MotAn is available at http://mouse.belozersky.msu.ru/motan.





    Categories: Journal Articles
  • Solution structure of the cyclic-nucleotide binding homology domain of a KCNH channel
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Qingxin Li , Hui Qi Ng , Ho Sup Yoon , Congbao Kang

    The carboxy-terminal region of the KCNH family of potassium channels contains a cyclic-nucleotide binding homology domain (CNBHD) that is important for channel gating and trafficking. The solution structure of the CNBHD of the KCNH potassium of zebrafish was determined using solution NMR spectroscopy. This domain exists as a monomer under solution conditions and adopts a similar fold to that determined by X-ray crystallography. The CNBHD does not bind cAMP because residue Y740 blocks the entry of cyclic-nucleotide to the binding pocket. Relaxation results show that the CNBHD is rigid except that some residues in the loop between β6 and β7 are flexible. Our results will be useful to understand the gating mechanism of KCNH family members through the CNBHD.





    Categories: Journal Articles
  • Structural diversity of a collagen-binding matrix protein from the byssus of blue mussels upon refolding
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Michael H. Suhre , Thomas Scheibel

    Blue mussels firmly adhere to a variety of different substrates by the byssus, an extracorporal structure consisting of several protein threads. These threads are mainly composed of fibrillar collagens called preCols which are embedded in a proteinaceous matrix. One of the two so far identified matrix proteins is the Proximal Thread Matrix Protein 1 (PTMP1). PTMP1 comprises two von Willebrand factor type A-like domains (A1 and A2) in a special arrangement. Here, we describe the refolding of recombinant PTMP1 from inclusion bodies. PTMP1 refolded into two distinct monomeric isoforms. Both isomers exhibited alternative intramolecular disulfide bonds. One of these isomers is thermodynamically favored and presumably represents the native form of PTMP1, while the other isoform is kinetically favored but is likely non-native. Oligomerization during refolding was influenced by, but not strictly dependent on disulfide formation. The conformational stability of PTMP1 indicates an influence of intramolecular disulfides on the native state, but not on unfolding intermediates. Monomeric PTMP1 exhibited a high thermal stability, dependent on the pH of the surrounding environment. Especially under acidic conditions the disulfide bonds were critically involved in thermal stability.





    Categories: Journal Articles
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