Journal of Structural Biology

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  • Histocompositional organization and toughening mechanisms in antler
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): John G. Skedros , Kendra E. Keenan , David M.L. Cooper , Roy D. Bloebaum

    Mechanical testing studies by Krauss et al. (2009) and Gupta et al. (2013) suggest that the extraordinary toughness of antler bone is primarily achieved by intrinsic/nanostructural mechanisms instead of extrinsic/microstructural mechanisms. However, this conclusion is based on data from extremely small specimens from one antler loaded only in tension, which impedes discernment of the relative importance of intrinsic vs. extrinsic mechanisms. In the present study we conducted analyses into the microstructural features of antler for details of potential additional microscale toughening characteristics, as suggested by recent mechanical testing studies of bulk specimens. The data are also considered in view of the above-mentioned studies concluding that extrinsic/microstructural toughening mechanisms are less important than nanoscale/intrinsic toughening mechanisms in antler. Mule deer antlers were evaluated using: (1) backscattered electron imaging for micro-mineralization, (2) circularly polarized light for osteonal interfacial complexity and collagen fiber orientation (CFO) heterogeneity, and (3) X-ray 3D micro-computed tomography for osteon/vessel orientation, density, and size. Results showed: (1) hyper-mineralized seams of approximately 3–4 microns thickness within relatively hypermineralized “zones” that course circuitously along osteonal interfaces, (2) highly heterogeneous CFO, including increased oblique-to-transverse CFO near/adjacent to osteon peripheries, and (3) osteons are often highly elongated in 2D. 3D reconstructions show that a considerable percentage of the vascular canals course obliquely with respect to the antler long axis. While results show multiple possible extrinsic-level histological characteristics in antler bone, it remains to be determined if microstructural characteristics become subsidiary to nanostructural characteristics in enhancing toughness during the majority of post-yield behavior of antler bone when loaded in a biologically relevant fashion.





    Categories: Journal Articles
  • Synchrotron X-ray micro-tomography imaging and analysis of wood degraded by Physisporinus vitreus and Xylaria longipes
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Marjan Sedighi Gilani , Matthieu N. Boone , Kevin Mader , Francis Willis Mathew Robert Schwarze

    Incubation of Norway spruce with Physisporinus vitreus and sycamore with Xylaria longipes results in reduction in density of these wood species that are traditionally used for the top and bottom plate of a violin, which follows by enhanced acoustic properties. We used Synchrotron X-ray micro-tomography, to study the three-dimensional structure of wood at the micro-scale level and the alterations of the density distribution after incubation with two white-rot fungi. Micro-tomography data from wood treated at different incubation periods are analyzed and compared with untreated (control) specimens to determine the wood density map and changes at the cell-wall level. Differences between the density of early- and latewood, xylem ray and around bordered pits in both Norway spruce and sycamore are studied. Three-dimensional hyphal networks of the P. vitreus and Xylaria longipes hyphae are visualized inside the cell lumina and their significance on the density of the early- and latewood cells after different incubation periods are discussed. The study illustrates the utility of X-ray micro-tomography for both qualitative and quantitative studies of a wide variety of biological systems and due to its high sensitivity, small structural changes can be quantified.





    Categories: Journal Articles
  • Ultrastructure and mineral composition of the cornea cuticle in the compound eyes of a supralittoral and a marine isopod
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Francisca I. Alagboso , Christian Reisecker , Sabine Hild , Andreas Ziegler

    The cuticle of the cornea in Crustacea is an interesting example of a composite material compromising between two distinct functions. As part of the dioptric apparatus of the ommatidia within the complex eye it forms transparent micro-lenses that should as well maintain the mechanical stability of the head capsule. We analyzed the ultrastructure and composition of the isopod cornea cuticle of the terrestrial species Ligia oceanica and the marine Sphaeroma serratum. We used a variety of tissue preparation methods, electron microscopic techniques as well as electron microprobe analysis and Raman spectroscopic imaging. The results reveal various structural adaptations that likely increase light transmission. These are an increase in the thickness of the epicuticle, a reduction of the thickness of the outer layer of calcite, a spatial restriction of pore canals to interommatidial regions, and, for S. serratum only, an increase in calcite crystal size. In both species protein–chitin fibrils within the proximal exocuticle form a peculiar reticular structure that does not occur within the cuticle of the head capsule. In L. oceanica differential mineralization results in a spherically shaped interface between mineralized and unmineralized endocuticle, likely an adaptation to increase the refractive power of the cornea maintaining the mechanical stability of the cuticle between the ommatidia. The results show that the habitat and differences in the general structure of the animal’s cuticle affect the way in which the cornea is adapted to its optical function.





    Categories: Journal Articles
  • X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Gabriel Demo , Veronika Papoušková , Jan Komárek , Pavel Kadeřávek , Olga Otrusinová , Pavel Srb , Alžbeta Rabatinová , Libor Krásný , Lukáš Žídek , Vladimír Sklenář , Michaela Wimmerová

    The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni2+ ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.





    Categories: Journal Articles
  • Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Naoyuki Miyazaki , Masatoshi Esaki , Teru Ogura , Kazuyoshi Murata

    Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.





    Categories: Journal Articles
  • Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin
    [Oct 2014]

    Publication date: August 2014
    Source:Journal of Structural Biology, Volume 187, Issue 2

    Author(s): Debiao Zhao , Xuejuan Wang , Junhui Peng , Chongyuan Wang , Fudong Li , Qianqian Sun , Yibo Zhang , Jiahai Zhang , Gang Cai , Xiaobing Zuo , Jihui Wu , Yunyu Shi , Zhiyong Zhang , Qingguo Gong

    c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807SH3a and D881SH3b are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.





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

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









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

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









    Categories: Journal Articles
  • Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies
    [Oct 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Guimei Yu , Frank Vago , Dongsheng Zhang , Jonathan E. Snyder , Rui Yan , Ci Zhang , Christopher Benjamin , Xi Jiang , Richard J. Kuhn , Philip Serwer , David H. Thompson , Wen Jiang

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged E scherichia coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.





    Categories: Journal Articles
  • M-free: Scoring the reference bias in sub-tomogram averaging and template matching
    [Oct 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Zhou Yu , Achilleas S. Frangakis

    Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.





    Categories: Journal Articles
  • Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections
    [Oct 2014]

    Publication date: Available online 6 October 2014
    Source:Journal of Structural Biology

    Author(s): Amélie Leforestier , Pierre Levitz , Thomas Préat , Peter Guttmann , Laurent J. Michot , Paul Tchenio

    Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40nm up to 2.5μm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy.





    Categories: Journal Articles
  • Efficient cryoprotection of macromolecular crystals using vapor diffusion of volatile alcohols
    [Oct 2014]

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

    Author(s): Christopher Farley , Douglas H. Juers

    Macromolecular X-ray crystallography, usually done at cryogenic temperature to limit radiation damage, often requires liquid cryoprotective soaking that can be labor intensive and damaging to crystals. Here we describe a method for cryoprotection that uses vapor diffusion of volatile cryoprotective agents into loop-mounted crystals. The crystal is mounted into a vial containing a small volume of an alcohol-based cryosolution. After a short incubation with the looped crystal sitting in the cryosolution vapor, the crystal is transferred directly from the vial into the cooling medium. Effective for several different protein crystals, the approach obviates the need for liquid soaking and opens up a heretofore underutilized class of cryoprotective agents for macromolecular crystallography.





    Categories: Journal Articles
  • Super-sampling SART with ordered subsets
    [Oct 2014]

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

    Author(s): Michael Kunz , Achilleas S. Frangakis

    In tomography, the quality of the reconstruction is essential because the complete cascade of the subsequent analysis is based on it. To date, weighted back-projection (WBP) has been the most commonly used technique due to its versatility and performance in sub-tomogram averaging. Here we present super-sampling SART that is based on the simultaneous algebraic reconstruction technique. While algebraic reconstruction techniques typically produce better contrast and lately showed a significant improvement in terms of processing speed, sub-tomogram averages derived from those reconstructions were inferior in resolution compared to those derived from WBP data. Super-sampling SART, however, outperforms both in term of contrast and the resolution achieved in sub-tomogram averaging several other tested methods and in particular WBP. The main feature of super-sampling SART, as the name implies, is the super-sampling option – by which parameter-based up-sampling and down-sampling are used to reduce artifacts. In particular, the aliasing that is omnipresent in the reconstruction can be practically eliminated without a significant increase in the computational time. Furthermore, super-sampling SART reaches convergence within a single iteration, making the processing time comparable to WBP, and eliminating the ambiguity of parameter-controlled convergence times. We find that grouping of projections increases the contrast, while when projections are used individually the resolution can be maximized. Using sub-tomogram averaging of ribosomes as a test case, we show that super-sampling SART achieves equal or better sub-tomogram averaging results than WBP, which is of particular importance in cryo-electron tomography.





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

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1









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

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1









    Categories: Journal Articles
  • Function-related adaptations of ultrastructure, mineral phase distribution and mechanical properties in the incisive cuticle of mandibles of Porcellio scaber Latreille, 1804
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Julia Huber , Helge-Otto Fabritius , Erika Griesshaber , Andreas Ziegler

    In terrestrial isopods the mandibles consist of a corpus carrying strong muscle tissue, and a pars incisiva (PI) that cuts dry leaves into smaller ingestible pieces. We studied the cuticle of the PI of Porcellio scaber in order to understand region-dependent differences in its ultrastructure, composition, and the resulting mechanical properties, employing several microscopic and analytical techniques as well as nanoindentation experiments. The cuticle of the incisive tip is not mineralized and consists of an unusually thick epicuticle containing thin fibrils, two subjacent cuticular layers, and a central core containing fibrils of different orientation, either longitudinal or circumferential. A thick epicuticle of the middle region just behind the tip projects long epicuticular extensions into the subjacent endocuticle, likely to prevent delamination. A distinct exocuticular layer is lacking in the middle region. Most chitin–protein fibrils within the endocuticle are oriented in parallel pointing towards the tip. Surprisingly, the middle region is mineralized by amorphous calcium phosphate (ACP) only. Near the base, ACP is successively replaced by amorphous calcium carbonate and calcite is restricted to a distal layer in the base. At the transition between middle and base, the epicuticle forms a hybrid material containing fibrils of the exocuticle. Nanoindentation experiments reveal an increase of the stiffness and hardness from the tip towards the base and significantly higher values on transversal in comparison to longitudinal planes. The results suggest that ultrastructure and composition are adapted for conveying high forces from a rather thin cutting edge to the stable base of the PI.





    Categories: Journal Articles
  • Repurposing TRASH: Emergence of the enzyme organomercurial lyase from a non-catalytic zinc finger scaffold
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Gurmeet Kaur , Srikrishna Subramanian

    The mercury resistance pathway enzyme organomercurial lyase (MerB) catalyzes the conversion of organomercurials to ionic mercury (Hg2+). Here, we provide evidence for the emergence of this enzyme from a TRASH-like, non-enzymatic, treble-clef zinc finger ancestor by domain duplication and fusion. Surprisingly, the structure-stabilizing metal-binding core of the treble-clef appears to have been repurposed in evolution to serve a catalytic role. Novel enzymatic functions are believed to have evolved from ancestral generalist catalytic scaffolds or from already specialized enzymes with catalytic promiscuity. The emergence of MerB from a zinc finger ancestor serves as a rare example of how a novel enzyme may emerge from a non-catalytic scaffold with a related binding function.





    Categories: Journal Articles
  • Crystal structure of PhoU from Pseudomonas aeruginosa, a negative regulator of the Pho regulon
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Sang Jae Lee , Ye Seol Park , Soon-Jong Kim , Bong-Jin Lee , Se Won Suh

    In Escherichia coli, seven genes (pstS, pstC, pstA, pstB, phoU, phoR, and phoB) are involved in sensing environmental phosphate (Pi) and controlling the expression of the Pho regulon. PhoU is a negative regulator of the Pi-signaling pathway and modulates Pi transport through Pi transporter proteins (PstS, PstC, PstA, and PstB) through the two-component system PhoR and PhoB. Inactivation of PhoY2, one of the two PhoU homologs in Mycobacterium tuberculosis, causes defects in persistence phenotypes and increased susceptibility to antibiotics and stresses. Despite the important biological role, the mechanism of PhoU function is still unknown. Here we have determined the crystal structure of PhoU from Pseudomonas aeruginosa. It exists as a dimer in the crystal, with each monomer consisting of two structurally similar three-helix bundles. Our equilibrium sedimentation measurements support the reversible monomer–dimer equilibrium model in which P. aeruginosa PhoU exists in solution predominantly as dimers, with monomers in a minor fraction, at low protein concentrations. The dissociation constant for PhoU dimerization is 3.2×10−6 M. The overall structure of P. aeruginosa PhoU dimer resembles those of Aquifex aeolicus PhoU and Thermotoga maritima PhoU2. However, it shows distinct structural features in some loops and the dimerization pattern.





    Categories: Journal Articles
  • Single virus detection by means of atomic force microscopy in combination with advanced image analysis
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Thomas Bocklitz , Evelyn Kämmer , Stephan Stöckel , Dana Cialla-May , Karina Weber , Roland Zell , Volker Deckert , Jürgen Popp

    In the present contribution virions of five different virus species, namely Varicella-zoster virus, Porcine teschovirus, Tobacco mosaic virus, Coliphage M13 and Enterobacteria phage PsP3, are investigated using atomic force microscopy (AFM). From the resulting height images quantitative features like maximal height, area and volume of the viruses could be extracted and compared to reference values. Subsequently, these features were accompanied by image moments, which quantify the morphology of the virions. Both types of features could be utilized for an automatic discrimination of the five virus species. The accuracy of this classification model was 96.8%. Thus, a virus detection on a single-particle level using AFM images is possible. Due to the application of advanced image analysis the morphology could be quantified and used for further analysis. Here, an automatic recognition by means of a classification model could be achieved in a reliable and objective manner.





    Categories: Journal Articles
  • Ocean acidification reduces the crystallographic control in juvenile mussel shells
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Susan C. Fitzer , Maggie Cusack , Vernon R. Phoenix , Nicholas A. Kamenos

    Global climate change threatens the oceans as anthropogenic carbon dioxide causes ocean acidification and reduced carbonate saturation. Future projections indicate under saturation of aragonite, and potentially calcite, in the oceans by 2100. Calcifying organisms are those most at risk from such ocean acidification, as carbonate is vital in the biomineralisation of their calcium carbonate protective shells. This study highlights the importance of multi-generational studies to investigate how marine organisms can potentially adapt to future projected global climate change. Mytilus edulis is an economically important marine calcifier vulnerable to decreasing carbonate saturation as their shells comprise two calcium carbonate polymorphs: aragonite and calcite. M. edulis specimens were cultured under current and projected pCO2 (380, 550, 750 and 1000μatm), following 6months of experimental culture, adults produced second generation juvenile mussels. Juvenile mussel shells were examined for structural and crystallographic orientation of aragonite and calcite. At 1000μatm pCO2, juvenile mussels spawned and grown under this high pCO2 do not produce aragonite which is more vulnerable to carbonate under-saturation than calcite. Calcite and aragonite were produced at 380, 550 and 750μatm pCO2. Electron back scatter diffraction analyses reveal less constraint in crystallographic orientation with increased pCO2. Shell formation is maintained, although the nacre crystals appear corroded and crystals are not so closely layered together. The differences in ultrastructure and crystallography in shells formed by juveniles spawned from adults in high pCO2 conditions may prove instrumental in their ability to survive ocean acidification.





    Categories: Journal Articles