Journal of Structural Biology

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  • The insertion domain 1 of class IIA dimeric glycyl-tRNA synthetase is a rubredoxin-like zinc ribbon
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Gurmeet Kaur , Srikrishna Subramanian

    The insertion domain 1 (ID1) of class IIA dimeric glycyl-tRNA synthetase (α2GRS) is an appended domain in the core catalytic region of the enzyme. ID1 has been shown to play a role in tRNA aminoacylation, mediating interaction with the acceptor arm of tRNA and diadenosine tetraphosphate (Ap4A) synthesis. Mutations in α2GRS, including those in the ID1 region, have been implicated in distal hereditary motor neuropathy-V (dHMN-V) and Charcot–Marie–Tooth (CMT) disease. Through sequence and structure based evolutionary analysis, we show that ID1 of α2GRS is a rubredoxin-like zinc ribbon domain. The zinc-chelating cysteines of ID1 are well conserved in all archaeal versions of the enzyme and also in several eukaryotes, which most likely have acquired them via horizontal gene transfer from bacteria; but in all other eukaryotes, the zinc-chelating residues are not preserved. ID1 from bacteria display a selective preservation of zinc-binding residues, ranging from complete conservation to complete loss. The ID1 from different organisms harbor variable-sized non-conserved insertions between the two zinc-binding half-sites of the zinc ribbon. Three of the previously identified CMT-associated mutations in α2GRS, viz., human D146N, mouse C157R and human S211F, are located in the zinc ribbon region of ID1. Interestingly, human Asp146 which is implicated in the synthesis of Ap4A, a molecule known to act during neuronal transmission, has also been reported to be mutated in dHMN-V, suggesting a possible link between hereditary motor neuropathy and Ap4A synthesis.





    Categories: Journal Articles
  • CapsidMaps: Protein–protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Mauricio Carrillo-Tripp , Daniel Jorge Montiel-García , Charles L. Brooks III , Vijay S. Reddy

    Structural analysis and visualization of protein–protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ–ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.





    Categories: Journal Articles
  • CNS myelin sheath is stochastically built by homotypic fusion of myelin membranes within the bounds of an oligodendrocyte process
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Sara Szuchet , Lauren L. Nielsen , Miriam S. Domowicz , Jotham R. Austin II , Dimitrios L. Arvanitis

    Myelin – the multilayer membrane that envelops axons – is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into “myelinophore organelles” in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease.





    Categories: Journal Articles
  • Changes in the micro- and nanostructure of siliceous valves in the diatom Synedra acus under the effect of colchicine treatment at different stages of the cell cycle
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Ksenia V. Kharitonenko , Yekaterina D. Bedoshvili , Yelena V. Likhoshway

    The important role of the cytoskeleton in the morphogenesis of siliceous frustule components, which are synthesized within the diatom cells, has been revealed due to experiments with microtubule inhibitors. It has been shown that colchicine entering the diatom cell inhibits polymerization of tubulin, the main protein of microtubules, thereby disrupting the normal processes of biogenic silica deposition and daughter valve morphogenesis. In this study, experiments with a synchronized culture of the pennate diatom Synedra acus have been performed to determine the timing and duration of the formation of various valve components and analyze the effect of colchicine at a subtoxic concentration on the structure of daughter valves at different stages of their morphogenesis. Electron microscopic analysis has revealed several types of micro- and nanoscale anomalies in daughter valve morphology, with their frequency varying depending on the time of colchicine treatment. Laser scanning microscopy of preparations vitally stained with Tubulin Tracker Green has shown that polymerized tubulin at early stages of valve morphogenesis is localized along the periphery of the developing valve. This is evidence for an important role of microtubules in the horizontal growth of the valve at the stage when its general structural pattern is established, including its shape and arrangement of basic micro- and nanostructures. Treatment with a microtubule inhibitor at a certain stage of valve morphogenesis makes it possible to obtain new forms with a specific structure of siliceous components that hold promise for use in nanotechnologies.





    Categories: Journal Articles
  • The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Barbara J. Tarasevich , John S. Philo , Nasib Karl Maluf , Susan Krueger , Garry W. Buchko , Genyao Lin , Wendy J. Shaw

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7–4.1, pH 4.5–8, 50mmol/L(mM) to 200mM NaCl, 0.065–2mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(−P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.





    Categories: Journal Articles
  • SubspaceEM: A fast maximum-a-posteriori algorithm for cryo-EM single particle reconstruction
    [Apr 2015]

    Publication date: Available online 31 March 2015
    Source:Journal of Structural Biology

    Author(s): Nicha C. Dvornek , Fred J. Sigworth , Hemant D. Tagare

    Single particle reconstruction methods based on the maximum-likelihood principle and the expectation–maximization (E–M) algorithm are popular because of their ability to produce high resolution structures. However, these algorithms are computationally very expensive, requiring a network of computational servers. To overcome this computational bottleneck, we propose a new mathematical framework for accelerating maximum-likelihood reconstructions. The speedup is by orders of magnitude and the proposed algorithm produces similar quality reconstructions compared to the standard maximum-likelihood formulation. Our approach uses subspace approximations of the cryo-electron microscopy (cryo-EM) data and projection images, greatly reducing the number of image transformations and comparisons that are computed. Experiments using simulated and actual cryo-EM data show that speedup in overall execution time compared to traditional maximum-likelihood reconstruction reaches factors of over 300.





    Categories: Journal Articles
  • The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity
    [Apr 2015]

    Publication date: Available online 27 March 2015
    Source:Journal of Structural Biology

    Author(s): Rubén Cebrián , Manuel Martínez-Bueno , Eva Valdivia , Armando Albert , Mercedes Maqueda , María José Sánchez-Barrena

    The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.





    Categories: Journal Articles
  • Dimeric WH2 repeats of VopF sequester actin monomers into non-nucleating linear string conformations: An X-ray scattering study
    [Apr 2015]

    Publication date: Available online 25 March 2015
    Source:Journal of Structural Biology

    Author(s): Balendu Sankara Avvaru , Julien Pernier , Marie-France Carlier

    VopF and VopL are highly similar virulence-factors of Vibrio cholerae and Vibrio parahaemolyticus respectively that disrupt the host’s actin cytoskeleton, using a unique organization in dimerized WH2 repeats. Association of dimerized WH2 domains with the barbed face of actin confers multifunctional activities to VopF in vitro, including G-actin sequestration and filament nucleation, barbed end tracking and uncapping. Here, small angle X-ray scattering (SAXS) measurements of complexes of VopF with actin and structural modeling reveal that VopF stabilizes linear actin-strings that differ from canonical actin filament architectures but represent non-polymerizable sequestered forms of actin. The results exclude that VopL binds the pointed end of actin filaments in the template filament nucleation mechanism derived from crystallographic studies.





    Categories: Journal Articles
  • Functionalization of biomineral reinforcement in crustacean cuticle: Calcite orientation in the partes incisivae of the mandibles of Porcellio scaber and the supralittoral species Tylos europaeus (Oniscidea, Isopoda)
    [Apr 2015]

    Publication date: Available online 25 March 2015
    Source:Journal of Structural Biology

    Author(s): Julia Huber , Erika Griesshaber , Fitriana Nindiyasari , Wolfgang W. Schmahl , Andreas Ziegler

    In arthropods the cuticle forms an exoskeleton with its physical and chemical properties adapted to functions of distinct skeletal elements. The cuticle of the partes incisivae (PI) in mandibles of terrestrial isopods is a composite of chitin-protein fibrils/fibres and minerals. It consists of an unmineralized tip, a middle region with organic fibrils reinforced mainly with amorphous calcium phosphate and a base region mineralized with amorphous calcium carbonate and calcite. In this study we extend our work on the structure and material properties of the incisive cuticle employing electron backscatter diffraction (EBSD), and investigate calcite orientation patterns in the PI of two terrestrial isopod species from different habitats. We trace small-scale differences in texture sharpness and calcite microstructure, and compare calcite organization and orientation patterns in the PI with those in the tergites of the same isopod species. We observe that in the PI calcite orientation, the degree of crystal alignment, and mode of crystalline domain assemblage is highly varied within short length scales. This contrasts to calcite organization in the tergite cuticle, where calcite has only one specific texture pattern. Such a large range in the variation of calcite organization has not been observed in other carbonate biological hard tissues, such as shells and teeth, where one specific texture and microstructure prevails. Thus, the investigated isopod species are able to control crystallization of the amorphous carbonate precursor in a differential way, most probably related to the function of the individual skeletal element and the animals’ behavior.





    Categories: Journal Articles
  • Structural insight into the oxidation of sinapic acid by CotA laccase
    [Apr 2015]

    Publication date: Available online 20 March 2015
    Source:Journal of Structural Biology

    Author(s): Tian Xie , Zhongchuan Liu , Qian Liu , Ganggang Wang

    Laccases can oxidize plenty of substrates by use of molecular oxygen as the final electron acceptor. The broad substrate spectrum is further expanded by using redox mediators in so-called laccase–mediator systems, but the structural studies on interactions between laccases and natural mediators are still absent. In this study, the crystal structure of CotA/sinapic acid complex is solved, structural comparison has revealed a novel substrate binding mode. The residue of His419 instead of His497 is bonding to the sinapic acid (SA) as the primary electron acceptor. Moreover, the binding of SA leads to 10° rotation on Arg416, our mutagenesis data exhibits that the residue Arg416 is crucial in the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and syringaldazine (SGZ). Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. By analyzing interactions between CotA and SA, it is indicated that the presence of methoxy groups in the ortho-position of the phenolic structure is crucial for the substrate recognition by CotA laccase. This work establishes structure–function relationships for laccase–natural mediator system.
    Graphical abstract




    Categories: Journal Articles
  • Crystal structures of S-adenosylhomocysteine hydrolase from the thermophilic bacterium Thermotoga maritima
    [Apr 2015]

    Publication date: Available online 17 March 2015
    Source:Journal of Structural Biology

    Author(s): Yingying Zheng , Chun-Chi Chen , Tzu-Ping Ko , Xiansha Xiao , Yunyun Yang , Chun-Hsiang Huang , Guojun Qian , Weilan Shao , Rey-Ting Guo

    S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD+ as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD+ show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD+-binding domain and the C-terminal domain. The NAD+ binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD+ binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD+ requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability.





    Categories: Journal Articles
  • Structural insights into specific crRNA G-rich sequence binding by Meiothermus ruber Cse2
    [Apr 2015]

    Publication date: Available online 16 March 2015
    Source:Journal of Structural Biology

    Author(s): Su Liu , Zuanning Yuan , Y. Adam Yuan

    CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-mediated defense against invading nucleic acids is a process recently discovered in prokaryotes, which includes recognition and incorporation of invading genetic elements, transcription and processing of CRISPR-RNA (crRNA) and targeting the invaders through base pair recognition. In the type I–E CRISPR–Cas system, Cse2 is proposed to provide a platform to facilitate the targeting of the invading dsDNA by crRNA. Here we report the crystal structure of Meiothermus ruber Cse2 at 2.8Å. M. ruber Cse2 adopts an α-helical bundle scaffold, harbors a positive surface for nucleic acid binding and a conserved dimer interface with strikingly low buried surface area. M. ruber Cse2 selectively binds to G-rich crRNA sequence, which is stripped off from the Cse2-crRNA and Cascade–crRNA complexes by ssDNA or dsDNA with complementary sequence. Stable M. ruber Cascade is readily formed by co-expression of M. ruber Cascade proteins together with G-rich crRNA in vitro. Docking of M. ruber Cse2 structures into the Escherichia coli Cascade Cryo-EM envelope reveals a curved elongated shallow groove for ssRNA binding, which adopts a similar dimer interface discovered by high-resolution crystal structure of Cse2 within E. Coli Cascade. Taken together, our data provides the structural insights into crRNA G-rich sequence recognition by M. ruber Cse2 and reveals the potential structural mechanism for M. ruber Cascade assembly and function.





    Categories: Journal Articles
  • Electron cryotomography of vitrified cells with a Volta phase plate
    [Apr 2015]

    Publication date: Available online 12 March 2015
    Source:Journal of Structural Biology

    Author(s): Yoshiyuki Fukuda , Ulrike Laugks , Vladan Lučić , Wolfgang Baumeister , Radostin Danev

    Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1–3nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (∼400nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-β-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow.





    Categories: Journal Articles
  • The apicomplexan glideosome and adhesins – Structures and function
    [Apr 2015]

    Publication date: Available online 9 March 2015
    Source:Journal of Structural Biology

    Author(s): Lauren E. Boucher , Jürgen Bosch

    The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen–host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein–protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3









    Categories: Journal Articles
  • Paper of the Year Award
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3









    Categories: Journal Articles
  • Paper of the Year Award
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3









    Categories: Journal Articles
  • Alignment of direct detection device micrographs using a robust Optical Flow approach
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3

    Author(s): Vahid Abrishami , Javier Vargas , Xueming Li , Yifan Cheng , Roberto Marabini , Carlos Óscar Sánchez Sorzano , José María Carazo

    The introduction of direct detection devices in cryo-EM has shown that specimens present beam-induced motion (BIM). Consequently, in this work, we develop a BIM correction method at the image level, resulting in an integrated image in which the in-plane BIM blurring is compensated prior to particle picking. The methodology is based on a robust Optical Flow (OF) approach that can efficiently correct for local movements in a rapid manner. The OF works particularly well if the BIM pattern presents a substantial degree of local movements, which occurs in our data sets for Falcon II data. However, for those cases in which the BIM pattern corresponds to global movements, we have found it advantageous to first run a global motion correction approach and to subsequently apply OF. Additionally, spatial analysis of the Optical Flow allows for quantitative analysis of the BIM pattern. The software that incorporates the new approach is available in XMIPP (http://xmipp.cnb.csic.es).





    Categories: Journal Articles
  • Xlink Analyzer: Software for analysis and visualization of cross-linking data in the context of three-dimensional structures
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3

    Author(s): Jan Kosinski , Alexander von Appen , Alessandro Ori , Kai Karius , Christoph W. Müller , Martin Beck

    Structural characterization of large multi-subunit protein complexes often requires integrating various experimental techniques. Cross-linking mass spectrometry (XL-MS) identifies proximal protein residues and thus is increasingly used to map protein interactions and determine the relative orientation of subunits within the structure of protein complexes. To fully adapt XL-MS as a structure characterization technique, we developed Xlink Analyzer, a software tool for visualization and analysis of XL-MS data in the context of the three-dimensional structures. Xlink Analyzer enables automatic visualization of cross-links, identifies cross-links violating spatial restraints, calculates violation statistics, maps chemically modified surfaces, and allows interactive manipulations that facilitate analysis of XL-MS data and aid designing new experiments. We demonstrate these features by mapping interaction sites within RNA polymerase I and the Rvb1/2 complex. Xlink Analyzer is implemented as a plugin to UCSF Chimera, a standard structural biology software tool, and thus enables seamless integration of XL-MS data with, e.g. fitting of X-ray structures to EM maps. Xlink Analyzer is available for download at http://www.beck.embl.de/XlinkAnalyzer.html.





    Categories: Journal Articles