Journal Cover Journal of Structural Biology
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   ISSN (Print) 1047-8477 - ISSN (Online) 1095-8657
   Published by Elsevier Homepage  [3120 journals]
  • Alpha repeat proteins (αRep) as expression and crystallization
           helpers
    • Authors: Anne Chevrel; Agnes Mesneau; Dyana Sanchez; Louisa Celma; Sophie Quevillon-Cheruel; Andrea Cavagnino; Sylvie Nessler; Ines Li de la Sierra-Gallay; Herman van Tilbeurgh; Philippe Minard; Marie Valerio-Lepiniec; Agathe Urvoas
      Pages: 88 - 99
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Anne Chevrel, Agnes Mesneau, Dyana Sanchez, Louisa Celma, Sophie Quevillon-Cheruel, Andrea Cavagnino, Sylvie Nessler, Ines Li de la Sierra-Gallay, Herman van Tilbeurgh, Philippe Minard, Marie Valerio-Lepiniec, Agathe Urvoas
      We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.
      Graphical abstract image

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.08.002
       
  • Elfin: An algorithm for the computational design of custom
           three-dimensional structures from modular repeat protein building blocks
    • Authors: Chun-Ting Yeh; TJ Brunette; David Baker; Simon McIntosh-Smith; Fabio Parmeggiani
      Pages: 100 - 107
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Chun-Ting Yeh, TJ Brunette, David Baker, Simon McIntosh-Smith, Fabio Parmeggiani
      Computational protein design methods have enabled the design of novel protein structures, but they are often still limited to small proteins and symmetric systems. To expand the size of designable proteins while controlling the overall structure, we developed Elfin, a genetic algorithm for the design of novel proteins with custom shapes using structural building blocks derived from experimentally verified repeat proteins. By combining building blocks with compatible interfaces, it is possible to rapidly build non-symmetric large structures (>1000 amino acids) that match three-dimensional geometric descriptions provided by the user. A run time of about 20min on a laptop computer for a 3000 amino acid structure makes Elfin accessible to users with limited computational resources. Protein structures with controlled geometry will allow the systematic study of the effect of spatial arrangement of enzymes and signaling molecules, and provide new scaffolds for functional nanomaterials.
      Graphical abstract image

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.09.001
       
  • Curvature of designed armadillo repeat proteins allows modular peptide
           binding
    • Authors: Simon Hansen; Patrick Ernst; Sebastian L.B. König; Christian Reichen; Christina Ewald; Daniel Nettels; Peer R.E. Mittl; Benjamin Schuler; Andreas Plückthun
      Pages: 108 - 117
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Simon Hansen, Patrick Ernst, Sebastian L.B. König, Christian Reichen, Christina Ewald, Daniel Nettels, Peer R.E. Mittl, Benjamin Schuler, Andreas Plückthun
      Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n =27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.08.009
       
  • Self-assembly of repeat proteins: Concepts and design of new interfaces
    • Authors: Daniel Sanchez-deAlcazar; Sara H. Mejias; Kevin Erazo; Begoña Sot; Aitziber L. Cortajarena
      Pages: 118 - 129
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Daniel Sanchez-deAlcazar, Sara H. Mejias, Kevin Erazo, Begoña Sot, Aitziber L. Cortajarena
      In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein–protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose. In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.09.002
       
  • Classification of β-hairpin repeat proteins
    • Authors: Daniel B. Roche; Phuong Do Viet; Anastasia Bakulina; Layla Hirsh; Silvio C.E. Tosatto; Andrey V. Kajava
      Pages: 130 - 138
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Daniel B. Roche, Phuong Do Viet, Anastasia Bakulina, Layla Hirsh, Silvio C.E. Tosatto, Andrey V. Kajava
      In recent years, a number of new protein structures that possess tandem repeats have emerged. Many of these proteins are comprised of tandem arrays of β-hairpins. Today, the amount and variety of the data on these β-hairpin repeat (BHR) structures have reached a level that requires detailed analysis and further classification. In this paper, we classified the BHR proteins, compared structures, sequences of repeat motifs, functions and distribution across the major taxonomic kingdoms of life and within organisms. As a result, we identified six different BHR folds in tandem repeat proteins of Class III (elongated structures) and one BHR fold (up-and-down β-barrel) in Class IV (“closed” structures). Our survey reveals the high incidence of the BHR proteins among bacteria and viruses and their possible relationship to the structures of amyloid fibrils. It indicates that BHR folds will be an attractive target for future structural studies, especially in the context of age-related amyloidosis and emerging infectious diseases. This work allowed us to update the RepeatsDB database, which contains annotated tandem repeat protein structures and to construct sequence profiles based on BHR structural alignments.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.10.001
       
  • Polyglutamine expansion diseases: More than simple repeats
    • Authors: Alexandra Silva; Ana Viana de Almeida; Sandra Macedo-Ribeiro
      Pages: 139 - 154
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Alexandra Silva, Ana Viana de Almeida, Sandra Macedo-Ribeiro
      Polyglutamine (polyQ) repeat-containing proteins are widespread in the human proteome but only nine of them are associated with highly incapacitating neurodegenerative disorders. The genetic expansion of the polyQ tract in disease-related proteins triggers a series of events resulting in neurodegeneration. The polyQ tract plays the leading role in the aggregation mechanism, but other elements modulate the aggregation propensity in the context of the full-length proteins, as implied by variations in the length of the polyQ tract required to trigger the onset of a given polyQ disease. Intrinsic features such as the presence of aggregation-prone regions (APRs) outside the polyQ segments and polyQ-flanking sequences, which synergistically participate in the aggregation process, are emerging for several disease-related proteins. The inherent polymorphic structure of polyQ stretches places the polyQ proteins in a central position in protein–protein interaction networks, where interacting partners may additionally shield APRs or reshape the aggregation course. Expansion of the polyQ tract perturbs the cellular homeostasis and contributes to neuronal failure by modulating protein–protein interactions and enhancing toxic oligomerization. Post-translational modifications further regulate self-assembly either by directly altering the intrinsic aggregation propensity of polyQ proteins, by modulating their interaction with different macromolecules or by modifying their withdrawal by the cell quality control machinery. Here we review the recent data on the multifaceted aggregation pathways of disease-related polyQ proteins, focusing on ataxin-3, the protein mutated in Machado-Joseph disease. Further mechanistic understanding of this network of events is crucial for the development of effective therapies for polyQ diseases.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.09.006
       
  • Top surface blade residues and the central channel water molecules are
           conserved in every repeat of the integrin-like β-propeller structures
    • Authors: Alexander Denesyuk; Konstantin Denessiouk; Mark S. Johnson
      Pages: 155 - 161
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Alexander Denesyuk, Konstantin Denessiouk, Mark S. Johnson
      An integrin-like β-propeller domain contains seven repeats of a four-stranded antiparallel β-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type β-propeller. Here, we show unique structural links that join different blades of the β-propeller structure, which together with the structural motif for a single blade are repeated in a β-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type β-propeller domain and two non-integrin type β-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.10.005
       
  • The repeat structure of two paralogous genes, Yersinia ruckeri invasin
           (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light
           on the evolution of adhesive capacities of a fish pathogen
    • Authors: Agnieszka Wrobel; Claudio Ottoni; Jack C. Leo; Snorre Gulla; Dirk Linke
      Pages: 171 - 183
      Abstract: Publication date: February 2018
      Source:Journal of Structural Biology, Volume 201, Issue 2
      Author(s): Agnieszka Wrobel, Claudio Ottoni, Jack C. Leo, Snorre Gulla, Dirk Linke
      Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2017.08.008
       
  • Rapid increase of near atomic resolution virus capsid structures
           determined by cryo-electron microscopy
    • Authors: Phuong T. Ho; Vijay S. Reddy
      Pages: 1 - 4
      Abstract: Publication date: January 2018
      Source:Journal of Structural Biology, Volume 201, Issue 1
      Author(s): Phuong T. Ho, Vijay S. Reddy
      The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (∼3.0 Å) and size (∼310.0 Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508 Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9 Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.10.007
       
  • Sup35NMp morphology evaluation on Au, Si, formvar and mica surfaces using
           AFM, SEM and TEM
    • Authors: P.A. Sokolov; S.A. Bondarev; M.V. Belousov; G.A. Zhouravleva; N.A. Kasyanenko
      Pages: 5 - 14
      Abstract: Publication date: January 2018
      Source:Journal of Structural Biology, Volume 201, Issue 1
      Author(s): P.A. Sokolov, S.A. Bondarev, M.V. Belousov, G.A. Zhouravleva, N.A. Kasyanenko
      Prion and some other incurable human neurodegenerative diseases are associated with misfolding of specific proteins, followed by the formation of amyloids. Despite the widespread usage of the transmission electron and of the atomic force microscopy for studing such amyloids, many related methodological issues still have not been studied until now. Here, we consider one of the first amyloids found in Saccharomyces cerevisiae yeast, i.e. Sup35NMp, to study the adsorption of monomeric protein and its fibrils on the surface of mica, silica, gold and on formvar film. Comparison of linear characteristics of these units calculated by processing of images obtained by the atomic force, transmission and scanning electron microscopy was carried out. The minimal number of measurements of fibril diameters to obtain the values in a given confidence interval were determined. We investigated the film formed by monomeric protein on mica surface, which veiled some morphology features of fibrils. Besides, we revealed that parts of the Sup35NMp excluded from the fibril core can form a wide “coat”. The length of the protein forming the core of the fibrils was estimated.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.10.006
       
  • Distinguishing signal from autofluorescence in cryogenic correlated light
           and electron microscopy of mammalian cells
    • Authors: Stephen D. Carter; Shrawan K. Mageswaran; Zachary J. Farino; João I. Mamede; Catherine M. Oikonomou; Thomas J. Hope; Zachary Freyberg; Grant J. Jensen
      Pages: 15 - 25
      Abstract: Publication date: January 2018
      Source:Journal of Structural Biology, Volume 201, Issue 1
      Author(s): Stephen D. Carter, Shrawan K. Mageswaran, Zachary J. Farino, João I. Mamede, Catherine M. Oikonomou, Thomas J. Hope, Zachary Freyberg, Grant J. Jensen
      In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.10.009
       
  • Crystallographic and enzymatic insights into the mechanisms of Mg-ADP
           inhibition in the A1 complex of the A1AO ATP synthase
    • Authors: Dhirendra Singh; Gerhard Grüber
      Pages: 26 - 35
      Abstract: Publication date: January 2018
      Source:Journal of Structural Biology, Volume 201, Issue 1
      Author(s): Dhirendra Singh, Gerhard Grüber
      F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A1AO ATP synthases (A3B3CDE2FG2 ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A3B3D- and A3B3DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.10.008
       
  • Rational design of new materials using recombinant structural proteins:
           Current state and future challenges
    • Authors: Tara D. Sutherland; Mickey G. Huson; Trevor D. Rapson
      Pages: 76 - 83
      Abstract: Publication date: January 2018
      Source:Journal of Structural Biology, Volume 201, Issue 1
      Author(s): Tara D. Sutherland, Mickey G. Huson, Trevor D. Rapson
      Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials.
      Graphical abstract image

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.10.012
       
  • Combining Rosetta with molecular dynamics (MD): a benchmark of the
           MD-based ensemble protein design
    • Authors: Jan Ludwiczak; Adam Jarmula; Stanislaw Dunin-Horkawicz
      Abstract: Publication date: Available online 14 February 2018
      Source:Journal of Structural Biology
      Author(s): Jan Ludwiczak, Adam Jarmula, Stanislaw Dunin-Horkawicz
      Computational protein design is a set of procedures for computing amino acid sequences that will fold into a specified structure. Rosetta Design, a commonly used software for protein design, allows for the effective identification of sequences compatible with a given backbone structure, while molecular dynamics (MD) simulations can thoroughly sample near-native conformations. We benchmarked a procedure in which Rosetta design is started on MD-derived structural ensembles and showed that such a combined approach generates 20 to 30% more diverse sequences than currently available methods with only a slight increase in computation time. Importantly, the increase in diversity is achieved without a loss in the quality of the designed sequences assessed by their resemblance to natural sequences. We demonstrate that the MD-based procedure is also applicable to de novo design tasks started from a backbone structures without any sequence information. In addition, we implemented a protocol that can be used to assess the stability of designed models and to select the best candidates for experimental validation. In sum our results demonstrate that the MD ensemble-based flexible backbone design can be a viable method for protein design, especially for tasks that require a large pool of diverse sequences.

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2018.02.004
       
  • Structure and electrochemistry of proteins harboring iron-sulfur clusters
           of different nuclearities. Part II. [4Fe-4S] and [3Fe-4S] iron-sulfur
           proteins
    • Authors: Piero Zanello
      Abstract: Publication date: Available online 8 February 2018
      Source:Journal of Structural Biology
      Author(s): Piero Zanello
      In the context of the plethora of proteins harboring iron-sulfur clusters we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur clusters (namely: {Fe(Cys)4}, {[Fe2S2](Cys)4}, {[Fe2S2](Cys)3(X)} (X = Asp, Arg, His), {[Fe2S2](Cys)2(His)2}, {[Fe3S4](Cys)3}, {[Fe4S4](Cys)4} and {[Fe4S4](Sγ Cys)3(nonthiolate ligand)} cores) and their synthetic analogs. More recently we are focussing on structure/electrochemistry of metalloproteins harboring iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] clusters, we now depict the state of art of proteins containing [4Fe-4S] and [3Fe-4S] clusters.
      Graphical abstract image

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2018.01.010
       
  • A complete structural characterization of the desferrioxamine E
           biosynthetic pathway from the fire blight pathogen Erwinia amylovora
    • Authors: Marco Salomone-Stagni; Joseph D. Bartho; Ivan Polsinelli; Dom Bellini; Martin A. Walsh; Nicola Demitri; Stefano Benini
      Abstract: Publication date: Available online 8 February 2018
      Source:Journal of Structural Biology
      Author(s): Marco Salomone-Stagni, Joseph D. Bartho, Ivan Polsinelli, Dom Bellini, Martin A. Walsh, Nicola Demitri, Stefano Benini
      The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43–47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).
      Graphical abstract image

      PubDate: 2018-02-14T23:58:16Z
      DOI: 10.1016/j.jsb.2018.02.002
       
  • Comparison of 3D cellular imaging techniques based on scanned electron
           probes: serial block face SEM vs. axial bright-field STEM tomography
    • Authors: E.L. McBride; A. Rao; G. Zhang; J.D. Hoyne; G.N. Calco; B.C. Kuo; Q. He; A.A. Prince; I.D. Pokrovskaya; B. Storrie; A.A. Sousa; M.A. Aronova; R.D. Leapman
      Abstract: Publication date: Available online 1 February 2018
      Source:Journal of Structural Biology
      Author(s): E.L. McBride, A. Rao, G. Zhang, J.D. Hoyne, G.N. Calco, B.C. Kuo, Q. He, A.A. Prince, I.D. Pokrovskaya, B. Storrie, A.A. Sousa, M.A. Aronova, R.D. Leapman
      Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼ 3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50 to 100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

      PubDate: 2018-02-03T03:45:24Z
      DOI: 10.1016/j.jsb.2018.01.012
       
  • Cep120 promotes microtubule formation through a unique tubulin binding C2
           domain
    • Authors: Ashwani Sharma; Samuel F. Gerard; Natacha Olieric; Michel O. Steinmetz
      Abstract: Publication date: Available online 1 February 2018
      Source:Journal of Structural Biology
      Author(s): Ashwani Sharma, Samuel F. Gerard, Natacha Olieric, Michel O. Steinmetz
      Centrioles are microtubule-based structures that play essential roles in cell division and cilia biogenesis. Cep120 is an important protein for correct centriole formation and mutations in the Cep120 gene cause severe human diseases like Joubert syndrome and complex ciliopathies. Here, we show that Cep120 contains three consecutive C2 domains that are followed by a coiled-coil dimerization domain. Surprisingly, unlike the classical C2 domains, all three Cep120 C2 domains lack calcium- and phospholipid-binding activities. However, biophysical and biochemical assays revealed that the N-terminal Cep120 C2 domain (C2A) binds to both tubulin and microtubules, and promotes microtubule formation. Structural analyses coupled with mutagenesis identified a highly conserved, positively charged residue patch on the surface of Cep120 C2A, which mediates the interaction with tubulin and microtubules. Together, our results establish Cep120 C2A as a unique microtubule-binding domain. They further provide insights into the molecular mechanism of Cep120 during centriole biogenesis.

      PubDate: 2018-02-03T03:45:24Z
      DOI: 10.1016/j.jsb.2018.01.009
       
  • Publisher Note
    • Abstract: Publication date: Available online 17 January 2018
      Source:Journal of Structural Biology


      PubDate: 2018-01-23T03:11:57Z
       
  • Volta phase plate data collection facilitates image processing and cryo-EM
           structure determination
    • Authors: Ottilie von Loeffelholz; Gabor Papai; Radostin Danev; Alexander G. Myasnikov; S. Kundhavai Natchiar; Isabelle Hazemann; Jean-François Ménétret; Bruno P. Klaholz
      Abstract: Publication date: Available online 11 January 2018
      Source:Journal of Structural Biology
      Author(s): Ottilie von Loeffelholz, Gabor Papai, Radostin Danev, Alexander G. Myasnikov, S. Kundhavai Natchiar, Isabelle Hazemann, Jean-François Ménétret, Bruno P. Klaholz
      A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The data were collected from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate (VPP) close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.003
       
  • Allosteric effects in bacteriophage HK97 procapsids revealed directly from
           covariance analysis of cryo EM data
    • Authors: Nan Xu; David Veesler; Peter C. Doerschuk; John E. Johnson
      Abstract: Publication date: Available online 10 January 2018
      Source:Journal of Structural Biology
      Author(s): Nan Xu, David Veesler, Peter C. Doerschuk, John E. Johnson
      The information content of cryo EM data sets exceeds that of the electron scattering potential (cryo EM) density initially derived for structure determination. Previously we demonstrated the power of data variance analysis for characterizing regions of cryo EM density that displayed functionally important variance anomalies associated with maturation cleavage events in Nudaurelia Omega Capensis Virus and the presence or absence of a maturation protease in bacteriophage HK97 procapsids. Here we extend the analysis in two ways. First, instead of imposing icosahedral symmetry on every particle in the data set during the variance analysis, we only assume that the data set as a whole has icosahedral symmetry. This change removes artifacts of high variance along icosahedral symmetry axes, but retains all of the features previously reported in the HK97 data set. Second we present a covariance analysis that reveals correlations in structural dynamics (variance) between the interior of the HK97 procapsid with the protease and regions of the exterior (not seen in the absence of the protease). The latter analysis corresponds well with hydrogen deuterium exchange studies previously published that reveal the same correlation.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.013
       
  • Structure of the Bacillus anthracis dTDP-l-rhamnose biosynthetic pathway
           enzyme: dTDP-α-d-glucose 4,6-dehydratase, RfbB
    • Authors: Trevor Gokey; Andrei S. Halavaty; George Minasov; Wayne F. Anderson; Misty L. Kuhn
      Abstract: Publication date: Available online 10 January 2018
      Source:Journal of Structural Biology
      Author(s): Trevor Gokey, Andrei S. Halavaty, George Minasov, Wayne F. Anderson, Misty L. Kuhn
      Many bacteria require l-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-l-rhamnose, which is produced by the dTDP-l-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-α-d-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.006
       
  • Impact of Intracellular Ionic Strength on Dimer Binding in the NF-kB
           Inducing Kinase
    • Authors: Michael R. Jones; Joshua Yue; Angela K. Wilson
      Abstract: Publication date: Available online 8 January 2018
      Source:Journal of Structural Biology
      Author(s): Michael R. Jones, Joshua Yue, Angela K. Wilson
      Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-kB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly.
      Graphical abstract image

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.004
       
  • Optimizing “Self-Wicking” Nanowire Grids
    • Authors: Hui Wei; Venkata P. Dandey; Zhening Zhang; Ashleigh Raczkowski; Willam J. Rice; Bridget Carragher; Clinton S. Potter
      Abstract: Publication date: Available online 6 January 2018
      Source:Journal of Structural Biology
      Author(s): Hui Wei, Venkata P. Dandey, Zhening Zhang, Ashleigh Raczkowski, Willam J. Rice, Bridget Carragher, Clinton S. Potter
      We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes. We explain how we characterize the quality of the grids and we describe their behavior under a variety of conditions.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.001
       
  • Comparative structural and enzymatic studies on Salmonella typhimurium
           diaminopropionate ammonia lyase reveal its unique features
    • Authors: G. Deka; S. Bisht; H.S. Savithri; M.R.N. Murthy
      Abstract: Publication date: Available online 30 December 2017
      Source:Journal of Structural Biology
      Author(s): G. Deka, S. Bisht, H.S. Savithri, M.R.N. Murthy
      Cellular metabolism of amino acids is controlled by a large number of pyridoxal 5′-phosphate (PLP) dependent enzymes. Diaminopropionate ammonia lyase (DAPAL), a fold type II PLP-dependent enzyme, degrades both the D and L forms of diaminopropionic acid (DAP) to pyruvate and ammonia. Earlier studies on the Escherichia coli DAPAL (EcDAPAL) had suggested that a disulfide bond located close to the active site may be crucial for maintaining the geometry of the substrate entry channel and the active site. In order to obtain further insights into the catalytic properties of DAPAL, structural and functional studies on Salmonella typhimurium DAPAL (StDAPAL) were initiated. The three-dimensional X-ray crystal structure of StDAPAL was determined at 2.5 Å resolution. As expected, the polypeptide fold and dimeric organization of StDAPAL is similar to those of EcDAPAL. A phosphate group was located in the active site of StDAPAL and expulsion of this phosphate is probably essential to bring Asp125 to a conformation suitable for proton abstraction from the substrate (D-DAP). The unique disulfide bond of EcDAPAL was absent in StDAPAL, although the enzyme displayed comparable catalytic activity. Site directed mutagenesis of the cysteine residues involved in disulfide bond formation in EcDAPAL followed by functional and biophysical studies further confirmed that the disulfide bond is not necessary either for substrate binding or for catalysis. The activity of StDAPAL but not EcDAPAL was enhanced by monovalent cations suggesting subtle differences in the active site geometries of these two closely related enzymes.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.012
       
  • Editorial for special issue “Proteins with tandem repeats: sequences,
           structures and functions”☆
    • Authors: Andrey V. Kajava; Silvio C.E. Tosatto
      Abstract: Publication date: Available online 30 December 2017
      Source:Journal of Structural Biology
      Author(s): Andrey V. Kajava, Silvio C.E. Tosatto


      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.011
       
  • A convolutional autoencoder approach for mining features in cellular
           electron cryo-tomograms and weakly supervised coarse segmentation
    • Authors: Xiangrui Zeng; Miguel Ricardo Leung; Tzviya Zeev-Ben-Mordehai; Min Xu
      Abstract: Publication date: Available online 28 December 2017
      Source:Journal of Structural Biology
      Author(s): Xiangrui Zeng, Miguel Ricardo Leung, Tzviya Zeev-Ben-Mordehai, Min Xu
      Cellular electron cryo-tomography enables the 3D visualization of cellular organization in the near-native state and at submolecular resolution. However, the contents of cellular tomograms are often complex, making it difficult to automatically isolate different in situ cellular components. In this paper, we propose a convolutional autoencoder-based unsupervised approach to provide a coarse grouping of 3D small subvolumes extracted from tomograms. We demonstrate that the autoencoder can be used for efficient and coarse characterization of features of macromolecular complexes and surfaces, such as membranes. In addition, the autoencoder can be used to detect non-cellular features related to sample preparation and data collection, such as carbon edges from the grid and tomogram boundaries. The autoencoder is also able to detect patterns that may indicate spatial interactions between cellular components. Furthermore, we demonstrate that our autoencoder can be used for weakly supervised semantic segmentation of cellular components, requiring a very small amount of manual annotation.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.015
       
  • A novel small molecule displays two different binding modes during
           inhibiting H1N1 influenza A virus neuraminidases
    • Authors: Shanshan Guan; Yan Xu; Yongbo Qiao; Ziyu Kuai; Mengdan Qian; Xiaoyu Jiang; Song Wang; Hao Zhang; Wei Kong; Yaming Shan
      Abstract: Publication date: Available online 28 December 2017
      Source:Journal of Structural Biology
      Author(s): Shanshan Guan, Yan Xu, Yongbo Qiao, Ziyu Kuai, Mengdan Qian, Xiaoyu Jiang, Song Wang, Hao Zhang, Wei Kong, Yaming Shan
      Neuraminidase (NA) inhibitors can suppress NA activity to block the release of progeny virions and are effective against influenza viruses. As potential anti-flu drugs with unique functions, NA inhibitors are greatly concerned by the worldwide scientists. It has been reported recently that one of the novel quindoline derivatives named 7a, could inhibit both A/Puerto Rico/8/34 (H1N1) NA (NAPR) and A/California/04/09 (H1N1) NA (NACA). However, potential structure differences in the active site could be easily detected between the NAPR and NACA according to the flexibilities of their 150-loops located catalytic site. And no obvious 150-cavity could be observed in NACA crystal structure. In order to explore whether 7a could trigger the inhibition against these two NAs in the same way, a serial molecular dynamics simulation approach were applied in this study. The results indicated that 7a could be adopted under a relatively extended pose in the active center of NAPR. While in NACA-7a complex, the derivate preferred to be recognized and located on the side of active center. Interestingly, the potential of 7a was also found to be able to change the flexibility of the 150-loop in NACA that is absent of 150-cavity. Furthermore, a 150-cavity-like architecture could be induced in the active site of NACA. The results of this study revealed two kinds of binding modes of this novel small molecule inhibitor against NAs that might provide a theoretical basis for proposing novel inhibition mechanism and developing future influenza A virus inhibitors.
      Graphical abstract image

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.014
       
  • The Donald L. D. Caspar Structural Biology Symposium at Florida State
           University
    • Authors: Piotr Fajer; Kenneth Taylor; Alexei Soares
      Pages: 199 - 201
      Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3
      Author(s): Piotr Fajer, Kenneth Taylor, Alexei Soares


      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.11.009
       
  • An adventure with Don
    • Authors: Richard Henderson
      Pages: 202 - 203
      Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3
      Author(s): Richard Henderson


      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.05.014
       
  • The fuzzy image
    • Authors: James Clarage
      Pages: 204 - 212
      Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3
      Author(s): James Clarage
      This article celebrates the variety of Don Caspar’s research interests, with particular focus on those scientific investigations beyond the structural biology of viruses for which he is often associated. These lesser known, seemingly backwater projects, allow us to build up a portrait, in both word and image, of this prolific and creative scientist. Exploration of his ideas will reveal a close connection to other structural thinkers and artists throughout history, most notably the 17th century astronomer Johannes Kepler.

      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.04.010
       
  • X-ray diffraction measurement of cosolvent accessible volume in
           rhombohedral insulin crystals
    • Authors: Alexei S. Soares; Donald L.D. Caspar
      Pages: 213 - 218
      Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3
      Author(s): Alexei S. Soares, Donald L.D. Caspar
      X-ray crystallographic measurement of the number of solvent electrons in the unit cell of a protein crystal equilibrated with aqueous solutions of different densities provides information about preferential hydration in the crystalline state. Room temperature and cryo-cooled rhombohedral insulin crystals were equilibrated with 1.2M trehalose to study the effect of lowered water activity. The native and trehalose soaked crystals were isomorphous and had similar structures. Including all the low resolution data, the amplitudes of the structure factors were put on an absolute scale (in units of electrons per asymmetric unit) by constraining the integrated number of electrons inside the envelope of the calculated protein density map to equal the number deduced from the atomic model. This procedure defines the value of F(000), the amplitude at the origin of the Fourier transform, which is equal to the total number of electrons in the asymmetric unit (i.e. protein plus solvent). Comparison of the F(000) values for three isomorphous pairs of room temperature insulin crystals, three with trehalose and three without trehalose, indicates that 75±12 electrons per asymmetric unit were added to the crystal solvent when soaked in 1.2M trehalose. If all the water in the crystal were available as solvent for the trehalose, 304 electrons would have been added. Thus, the co-solvent accessible volume is one quarter of the total water in the crystal. Determination of the total number of electrons in a protein crystal is an essential first step for mapping the average density distribution of the disordered solvent.

      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.08.004
       
  • High resolution crystal structures of Clostridium botulinum neurotoxin A3
           and A4 binding domains
    • Authors: Jonathan R. Davies; Jay Rees; Sai Man Liu; K. Ravi Acharya
      Abstract: Publication date: Available online 26 December 2017
      Source:Journal of Structural Biology
      Author(s): Jonathan Davies, Jay Rees, Sai Man Liu, K. Ravi Acharya
      Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Å and 1.34 Å resolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.

      PubDate: 2017-12-27T04:45:50Z
      DOI: 10.1016/j.jsb.2017.12.010
       
  • Structural Characterization of SpoIIIAB Sporulation-essential Protein in
           Bacillus subtilis
    • Authors: N. Zeytuni; K.A. Flanagan; L.J. Worrall; S.C. Massoni; A.H. Camp; N.C.J. Strynadka
      Abstract: Publication date: Available online 26 December 2017
      Source:Journal of Structural Biology
      Author(s): N. Zeytuni, K.A. Flanagan, L.J. Worrall, S.C. Massoni, A.H. Camp, N.C.J. Strynadka
      Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Å resolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.

      PubDate: 2017-12-27T04:45:50Z
      DOI: 10.1016/j.jsb.2017.12.009
       
  • Crystal Structure of cystathionine β-synthase from honeybee Apis
           mellifera
    • Authors: Paula Giménez-Mascarell; Tomas Majtan; Iker Oyenarte; June Ereño-Orbea; Juraj Majtan; Jaroslav Klaudiny; Jan P. Kraus; Luis Alfonso Martínez-Cruz
      Abstract: Publication date: Available online 21 December 2017
      Source:Journal of Structural Biology
      Author(s): Paula Giménez-Mascarell, Tomas Majtan, Iker Oyenarte, June Ereño-Orbea, Juraj Majtan, Jaroslav Klaudiny, Jan P. Kraus, Luis Alfonso Martínez-Cruz
      Cystathionine β-synthase (CBS), the key enzyme in the transsulfuration pathway, links methionine metabolism to the biosynthesis of cellular redox controlling molecules. CBS catalyzes the pyridoxal-5’-phosphate-dependent condensation of serine and homocysteine to form cystathionine, which is subsequently converted into cysteine. Besides maintaining cellular sulfur amino acid homeostasis, CBS also catalyzes multiple hydrogen sulfide-generating reactions using cysteine and homocysteine as substrates. In mammals, CBS is activated by S-adenosylmethionine (AdoMet), where it can adopt two different conformations (basal and activated), but exists as a unique highly active species in fruit fly Drosophila melanogaster. Here we present the crystal structure of CBS from honeybey Apis mellifera, which shows a constitutively active dimeric species and let explain why the enzyme is not allosterically regulated by AdoMet. In addition, comparison of available CBS structures unveils a substrate-induced closure of the catalytic cavity, which in humans is affected by the AdoMet-dependent regulation and likely impaired by the homocystinuria causing mutation T191M.

      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.12.008
       
  • Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct
           kinetics between adipose tissue-derived mesenchymal stem cells and a
           mesenchymal cancer cell lineage.
    • Authors: Camila Cristina Fraga Faraco; Jerusa Araújo Quintão Arantes Faria; Marianna Kunrath Lima; Marcelo Coutinho de Miranda; Mariane Izabella Abreu de Melo; Andrea da Fonseca Ferreira; Michele Angela Rodrigues; Dawidson Assis Gomes
      Abstract: Publication date: Available online 19 December 2017
      Source:Journal of Structural Biology
      Author(s): Camila Cristina Fraga Faraco, Jerusa Araújo Quintão Arantes Faria, Marianna Kunrath Lima, Marcelo Coutinho de Miranda, Mariane Izabella Abreu de Melo, Andrea da Fonseca Ferreira, Michele Angela Rodrigues, Dawidson Assis Gomes
      Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 minutes of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 minutes of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 minutes in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 minutes of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.12.007
       
  • STEM tomography analysis of the trypanosome transition zone
    • Authors: Sylvain Trépout; Anne-Marie Tassin; Sergio Marco; Philippe Bastin
      Abstract: Publication date: Available online 14 December 2017
      Source:Journal of Structural Biology
      Author(s): Sylvain Trépout, Anne-Marie Tassin, Sergio Marco, Philippe Bastin
      The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9 + 0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.

      PubDate: 2017-12-22T04:19:44Z
      DOI: 10.1016/j.jsb.2017.12.005
       
  • Cover 2 - Editorial Board
    • Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3


      PubDate: 2017-12-22T04:19:44Z
       
  • Table of Contents / barcode
    • Abstract: Publication date: December 2017
      Source:Journal of Structural Biology, Volume 200, Issue 3


      PubDate: 2017-12-22T04:19:44Z
       
  • Structural characterization of ribT from Bacillus subtilis reveals it as a
           GCN5-related N-acetyltransferase
    • Authors: Ritika Srivastava; Amanpreet Kaur; Charu Sharma; Subramanian Karthikeyan
      Abstract: Publication date: Available online 11 December 2017
      Source:Journal of Structural Biology
      Author(s): Ritika Srivastava, Amanpreet Kaur, Charu Sharma, Subramanian Karthikeyan
      In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis.

      PubDate: 2017-12-12T03:55:25Z
      DOI: 10.1016/j.jsb.2017.12.006
       
  • Structural changes of homodimers in the PDB
    • Authors: Ryotaro Koike; Takayuki Amemiya; Tatsuya Horii; Motonori Ota
      Abstract: Publication date: Available online 9 December 2017
      Source:Journal of Structural Biology
      Author(s): Ryotaro Koike, Takayuki Amemiya, Tatsuya Horii, Motonori Ota
      Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes.

      PubDate: 2017-12-12T03:55:25Z
      DOI: 10.1016/j.jsb.2017.12.004
       
  • Correlative imaging reveals physiochemical heterogeneity of
           microcalcifications in human breast carcinomas
    • Authors: Jennie A.M.R. Kunitake; Siyoung Choi; Kayla X. Nguyen; Meredith M. Lee; Frank He; Daniel Sudilovsky; Patrick G. Morris; Maxine S. Jochelson; Clifford A. Hudis; David A. Muller; Peter Fratzl; Claudia Fischbach; Admir Masic; Lara A. Estroff
      Abstract: Publication date: Available online 6 December 2017
      Source:Journal of Structural Biology
      Author(s): Jennie A.M.R. Kunitake, Siyoung Choi, Kayla X. Nguyen, Meredith M. Lee, Frank He, Daniel Sudilovsky, Patrick G. Morris, Maxine S. Jochelson, Clifford A. Hudis, David A. Muller, Peter Fratzl, Claudia Fischbach, Admir Masic, Lara A. Estroff
      Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.
      Graphical abstract image

      PubDate: 2017-12-09T03:48:04Z
      DOI: 10.1016/j.jsb.2017.12.002
       
  • Comparative mechanical unfolding studies of spectrin domains R15, R16 and
           R17
    • Authors: Anna V. Glyakina; Ilya V. Likhachev; Nikolay K. Balabaev; Oxana V. Galzitskaya
      Abstract: Publication date: Available online 5 December 2017
      Source:Journal of Structural Biology
      Author(s): Anna V. Glyakina, Ilya V. Likhachev, Nikolay K. Balabaev, Oxana V. Galzitskaya
      Spectrins belong to repetitive three-helix bundle proteins that have vital functions in multicellular organisms and are of potential value in nanotechnology. To reveal the unique physical features of repeat proteins we have studied the structural and mechanical properties of three repeats of chicken brain α-spectrin (R15, R16 and R17) at the atomic level under stretching at constant velocities (0.01, 0.05 and 0.1Å·ps–1) and constant forces (700 and900pN) using molecular dynamics (MD) simulations at T = 300K. 114 independent MD simulations were performed and their analysis has been done. Despite structural similarity of these domains we have found that R15 is less mechanically stable than R16, which is less stable than R17. This result is in agreement with the thermal unfolding rates. Moreover, we have observed the relationship between mechanical stability, flexibility of the domains and the number of aromatic residues involved in aromatic clusters.

      PubDate: 2017-12-09T03:48:04Z
      DOI: 10.1016/j.jsb.2017.12.003
       
  • Photosome membranes merge and organize tending towards rhombohedral
           symmetry when light is emitted
    • Authors: Malika Ouldali; Virginie Maury; Gisèle Nicolas; Jean Lepault
      Abstract: Publication date: Available online 5 December 2017
      Source:Journal of Structural Biology
      Author(s): Malika Ouldali, Virginie Maury, Gisèle Nicolas, Jean Lepault
      Polynoid worm elytra emit light when mechanically or electrically stimulated. Specialized cells, the photocytes, contain light emitting machineries, the photosomes. Successive stimulations induce light intensity variations and show a coupling within and between photosomes. Here, we describe, using electron tomography of cryo-substituted elytra and freeze-fracturing, the structural transition associated to light emission: undulating tubules come closer, organize and their number forming photosomes increases. Two repeating undulating tubules in opposite phase compose the photosome. Undulations are located on three hexagonal layers that regularly repeat and are equally displaced, in x y and z. The tubule membranes within layers merge giving rise to rings that tend to obey to quasi-rhombohedral symmetry. Merging may result either from close-association, hemifusion (one leaflet fusion) or from fusion (two leaflets fusion). Although the resolution of tomograms is not sufficient to distinguish these three cases, freeze-fracturing shows that hemifusion is a frequent process that leads to an reversible anastomosed membrane complex favoring communications, appearing as a major coupling factor of photosome light emission.
      Graphical abstract image

      PubDate: 2017-12-09T03:48:04Z
      DOI: 10.1016/j.jsb.2017.12.001
       
  • Phylogenetic Analysis Predicts Structural Divergence for Proteobacterial
           ClpC Proteins
    • Authors: Justin M. Miller; Hamza Chaudhary; Justin D. Marsee
      Abstract: Publication date: Available online 10 November 2017
      Source:Journal of Structural Biology
      Author(s): Justin M. Miller, Hamza Chaudhary, Justin D. Marsee
      Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA, clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here.
      Graphical abstract image

      PubDate: 2017-11-11T16:39:34Z
      DOI: 10.1016/j.jsb.2017.11.003
       
  • Marker-free method for accurate alignment between correlated light,
           cryo-light, and electron cryo-microscopy data using sample support
           features
    • Authors: Karen Anderson; Christopher Mark Swift Dorit Hanein Niels Volkmann
      Abstract: Publication date: Available online 4 November 2017
      Source:Journal of Structural Biology
      Author(s): Karen L. Anderson, Christopher Page, Mark F. Swift, Dorit Hanein, Niels Volkmann
      Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

      PubDate: 2017-11-05T16:10:31Z
       
  • High-vacuum Optical Platform for cryo-CLEM (HOPE): a New Solution for
           Non-integrated Multiscale Correlative Light and Electron Microscopy
    • Authors: Shuoguo Li; Gang Ji; Yang Shi; Lasse Hyldgaard Klausen; Tongxin Niu; Shengliu Wang; Xiaojun Huang; Wei Ding; Xiang Zhang; Mingdong Dong; Wei Xu; Fei Sun
      Abstract: Publication date: Available online 4 November 2017
      Source:Journal of Structural Biology
      Author(s): Shuoguo Li, Gang Ji, Yang Shi, Lasse Hyldgaard Klausen, Tongxin Niu, Shengliu Wang, Xiaojun Huang, Wei Ding, Xiang Zhang, Mingdong Dong, Wei Xu, Fei Sun
      Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.

      PubDate: 2017-11-05T16:10:31Z
      DOI: 10.1016/j.jsb.2017.11.002
       
  • Influence of Physical Activity on Tibial Bone Material Properties in
           Laying Hens
    • Authors: A.B. Rodriguez-Navarro; H.M. McCormack; R.H. Fleming; P. Alvarez-Lloret; J. Romero-Pastor; N. Dominguez-Gasca; Tanya Prozorov; I.C. Dunn
      Abstract: Publication date: Available online 3 November 2017
      Source:Journal of Structural Biology
      Author(s): A.B. Rodriguez-Navarro, H.M. McCormack, R.H. Fleming, P. Alvarez-Lloret, J. Romero-Pastor, N. Dominguez-Gasca, Tanya Prozorov, I.C. Dunn
      Laying hens develop a type of osteoporosis that arises from a loss of structural bone, resulting in high incidence of fractures. In this study, a comparison of bone material properties was made for lines of hens created by divergent selection to have high and low bone strength and housed in either individual cages, with restricted mobility, or in an aviary system, with opportunity for increased mobility. Improvement of bone biomechanics in the high line hens and in aviary housing was mainly due to increased bone mass, thicker cortical bone and more medullary bone. However, bone material properties such as cortical and medullary bone mineral composition and crystallinity as well as collagen maturity did not differ between lines. However, bone material properties of birds from the different type of housing were markedly different. The cortical bone in aviary birds had a lower degree of mineralization and bone mineral was less mature and less organized than in caged birds. These differences can be explained by increased bone turnover rates due to the higher physical activity of aviary birds that stimulates bone formation and bone remodeling. Multivariate statistical analyses shows that both cortical and medullary bone contribute to breaking strengthThe cortical thickness was the single most important contributor while its degree of mineralization and porosity had a smaller contribution. Bone properties had poorer correlations with mechanical properties in cage birds than in aviary birds presumably due to the greater number of structural defects of cortical bone in cage birds.

      PubDate: 2017-11-05T16:10:31Z
      DOI: 10.1016/j.jsb.2017.10.011
       
 
 
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