Journal of Structural Biology

ScienceDirect RSS
  • 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
  • Nanoscale Three-Dimensional Imaging of the Human Myocyte
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Structural Biology

    Author(s): Matthew S. Sulkin , Fei Yang , Katherine M. Holzem , Brandon Van Leer , Cliff Bugge , Jacob I. Laughner , Karen Green , Igor R. Efimov

    The ventricular human myocyte is spatially organized for optimal ATP and Ca2+ delivery to sarcomeric myosin and ionic pumps during every excitation-contraction cycle. Comprehension of three-dimensional geometry of the tightly packed ultrastructure has been derived from discontinuous two-dimensional images, but has never been precisely reconstructed or analyzed in human myocardium. Using a focused ion beam scanning electron microscope, we created nanoscale resolution serial images to quantify the three-dimensional ultrastructure of a human left ventricular myocyte. Transverse tubules (t-tubule), lipid droplets, A-bands, and mitochondria occupy 1.8, 1.9, 10.8, and 27.9% of the myocyte volume, respectively. The complex t-tubule system has a small tortuosity (1.04 ± 0.01), and is composed of long transverse segments with diameters of 317 ± 24 nm and short branches. Our data indicates that lipid droplets located well beneath the sarcolemma are proximal to t-tubules, where 59% (13 of 22) of lipid droplet centroids are within 0.50 μm of a t-tubule. This spatial association could have an important implication in the development and treatment of heart failure because it connects two independently known pathophysiological alterations, a substrate switch from fatty acids to glucose and t-tubular derangement.





    Categories: Journal Articles
  • 2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins
    [Aug 2014]

    Publication date: Available online 23 August 2014
    Source:Journal of Structural Biology

    Author(s): Jean-Marc Jeckelmann , Daniel Harder , Zöhre Ucurum , Dimitrios Fotiadis

    Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively-stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2 Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.





    Categories: Journal Articles
  • Dynamics of bovine opsin bound to G-protein fragments
    [Aug 2014]

    Publication date: Available online 17 August 2014
    Source:Journal of Structural Biology

    Author(s): Minoru Sugihara , Makiko Suwa , Ana-Nicoleta Bondar

    G protein-coupled receptors (GPCRs) are a large class of membrane proteins that mediate communication of the cell with the outer environment. Upon activation by an agonist, GPCRs undergo large-scale conformational changes that enable binding of the G protein to the receptor. A key open question concerns the mechanism of the long-distance coupling between the agonist-binding site and the cytoplasmic site where G protein binds. Here we address this question by exploring the molecular dynamics of bovine opsin bound to three different fragments of G-proteins. We find that an extended network of hydrogen bonds connects the agonist retinal binding site to the G protein binding site via conserved amino acid residues. The dynamics of the hydrogen-bonding network inside opsin couples to interactions at the G protein binding site.





    Categories: Journal Articles
  • Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria
    [Aug 2014]

    Publication date: Available online 7 August 2014
    Source:Journal of Structural Biology

    Author(s): Asfarul S. Haque , Ketan D. Patel , Mandar V. Deshmukh , Arush Chhabra , Rajesh S. Gokhale , Rajan Sankaranarayanan

    Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order ‘bi–bi’ reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4′-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi–bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3–4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation–π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi–bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.





    Categories: Journal Articles
  • Crystal structures of Ophiostoma piceae sterol esterase: Structural insights into activation mechanism and product release
    [Aug 2014]

    Publication date: Available online 7 August 2014
    Source:Journal of Structural Biology

    Author(s): Javier Gutiérrez-Fernández , María Eugenia Vaquero , Alicia Prieto , Jorge Barriuso , María Jesús Martínez , Juan A. Hermoso

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16–α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.





    Categories: Journal Articles
  • Crystal structure of kiwellin, a major cell-wall protein from kiwifruit
    [Aug 2014]

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

    Author(s): Cyril Hamiaux , Ratnasiri Maddumage , Martin J. Middleditch , Roneel Prakash , David A. Brummell , Edward N. Baker , Ross G. Atkinson

    Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05Å resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi β-barrel fold hooked to an N-terminal β hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.





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

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









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

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









    Categories: Journal Articles
  • Structure of the 3.3MDa, in vitro assembled, hubless bacteriophage T4 baseplate
    [Aug 2014]

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

    Author(s): Moh Lan Yap , Thomas Klose , Pavel Plevka , Anastasia Aksyuk , Xinzheng Zhang , Fumio Arisaka , Michael G. Rossmann

    The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an E scherichia coli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5MDa baseplate changes its structure from a “dome” shape to a “star” shape. An in vitro assembled hubless baseplate has been crystallized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6.





    Categories: Journal Articles
  • Quantifying resolution limiting factors in subtomogram averaged cryo-electron tomography using simulations
    [Aug 2014]

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

    Author(s): Lenard M. Voortman , Miloš Vulović , Massimiliano Maletta , Andreas Voigt , Erik M. Franken , Angelita Simonetti , Peter J. Peters , Lucas J. van Vliet , Bernd Rieger

    Cryo-electron tomography (CET) is the only available technique capable of characterizing the structure of biological macromolecules in conditions close to the native state. With the advent of subtomogram averaging, as a post-processing step to CET, resolutions in the (sub-) nanometer range have become within reach. In addition to advances in instrumentation and experiments, the reconstruction scheme has improved by inclusion of more accurate contrast transfer function (CTF) correction methods, better defocus estimation, and better alignments of the tilt-series and subtomograms. To quantify the importance of each contribution, we have split the full process from data collection to reconstruction into different steps. For the purpose of evaluation we have acquired tilt-series of ribosomes in such a way that we could precisely determine the defocus of each macromolecule. Then, we simulated tilt-series using the InSilicoTEM package and applied tomogram reconstruction and subtomogram averaging. Through large scale simulations under different conditions and parameter settings we find that tilt-series alignment is the resolution limiting factor for our experimental data. Using simulations, we find that when this alignment inaccuracy is alleviated, tilted CTF correction improves the final resolution, or equivalently, the same resolution can be achieved using less particles. Furthermore, we predict from which resolution onwards better CTF correction and defocus estimation methods are required. We obtain a final average using 3198 ribosomes with a resolution of 2.2nm on the experimental data. Our simulations suggest that with the same number of particles a resolution of 1.2nm could be achieved by improving the tilt-series alignment.





    Categories: Journal Articles