Journal of Structural Biology

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  • Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    Author(s): Caroline Kizilyaprak , Giovanni Longo , Jean Daraspe , Bruno M. Humbel

    In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼500nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB–SEM tomography, became available to image much larger volumes. During TEM tomography and FIB–SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.





    Categories: Journal Articles
  • Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    Author(s): Jose-Ignacio Agulleiro , Jose-Jesus Fernandez

    Tomo3D is a program for fast tomographic reconstruction on multicore computers. Its high speed stems from code optimization, vectorization with Streaming SIMD Extensions (SSE), multithreading and optimization of disk access. Recently, Advanced Vector eXtensions (AVX) have been introduced in the x86 processor architecture. Compared to SSE, AVX double the number of simultaneous operations, thus pointing to a potential twofold gain in speed. However, in practice, achieving this potential is extremely difficult. Here, we provide a technical description and an assessment of the optimizations included in Tomo3D to take advantage of AVX instructions. Tomo3D 2.0 allows huge reconstructions to be calculated in standard computers in a matter of minutes. Thus, it will be a valuable tool for electron tomography studies with increasing resolution needs.





    Categories: Journal Articles
  • Use of a “silver bullet” to resolve crystal lattice dislocation disorder: A cobalamin complex of Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    Author(s): Thomas Lagautriere , Ghader Bashiri , Edward N. Baker

    The use of small molecules as “silver bullets” that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order–disorder phenomenon that complicated the structural analysis of the enzyme Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order–disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.





    Categories: Journal Articles
  • Crystal structure of a periplasmic solute binding protein in metal-free, intermediate and metal-bound states from Candidatus Liberibacter asiaticus
    [Feb 2015]

    Publication date: Available online 30 January 2015
    Source:Journal of Structural Biology

    Author(s): Nidhi Sharma , Purushotham Selvakumar , Sumit Bhose , Dilip Kumar Ghosh , Pravindra Kumar , Ashwani Kumar Sharma

    The Znu system, a member of ABC transporter family, is critical for survival and pathogenesis of Candidatus Liberibacter asiaticus (CLA). Two homologues of this system have been identified in CLA. Here, we report high resolution crystal structure of a periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) in metal-free, intermediate and metal-bound states. CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific solute binding proteins (SBPs) of cluster A-I family. The overall fold of CLas-ZnuA2 is similar to the related cluster A-I family SBPs. The sequence and structure analysis revealed the unique features of CLas-ZnuA2. The comparison of CLas-ZnuA2 structure in three states showed that metal binding and release is facilitated by a large displacement along with a change in orientation of the side chain for one of the metal binding residue (His39) flipped away from metal binding site in metal-free form. The crystal structure captured in intermediate state of metal binding revealed the changes in conformation and interaction of the loop hosting His39 during the metal binding. A rigid body movement of C-domain along with partial unfolding of linker helix at its C-terminal during metal binding, as reported for PsaA, was not observed in CLas-ZnuA2. The present results suggest that despite showing maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance of CLas-ZnuA2 seems to be closer to Zn-specific SBPs of cluster A-I family.





    Categories: Journal Articles
  • A statistical approach to the initial volume problem in Single Particle Analysis by Electron Microscopy
    [Feb 2015]

    Publication date: Available online 28 January 2015
    Source:Journal of Structural Biology

    Author(s): C.O.S. Sorzano , J. Vargas , J.M. de la Rosa-Trevín , J. Otón , A.L. Álvarez-Cabrera , V. Abrishami , E. Sesmero , R. Marabini , J.M. Carazo

    Cryo Electron Microscopy is a powerful Structural Biology technique, allowing the elucidation of the three-dimensional structure of biological macromolecules. In particular, the structural study of purified macromolecules –often referred as Single Particle Analysis(SPA)– is normally performed through an iterative process that needs a first estimation of the three-dimensional structure that is progressively refined using experimental data. It is well-known the local optimisation nature of this refinement, so that the initial choice of this first structure may substantially change the final result. Computational algorithms aiming to providing this first structure already exist. However, the question is far from settled and more robust algorithms are still needed so that the refinement process can be performed with sufficient guarantees. In this article we present a new algorithm that addresses the initial volume problem in SPA by setting it in a Weighted Least Squares framework and calculating the weights through a statistical approach based on the cumulative density function of different image similarity measures. We show that the new algorithm is significantly more robust than other state-of-the-art algorithms currently in use in the field. The algorithm is available as part of the software suite Xmipp (http://xmipp.cnb.csic.es) and Scipion (http://scipion.cnb.csic.es) under the name “Significant”.





    Categories: Journal Articles
  • Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy
    [Feb 2015]

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

    Author(s): Zahra El-Schich , Anna Mölder , Helena Tassidis , Pirkko Härkönen , Maria Falck Miniotis , Anette Gjörloff Wingren

    We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1–3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.





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

    Publication date: Available online 22 January 2015
    Source:Journal of Structural Biology

    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
  • Cryo-electron tomography of plunge-frozen whole bacteria and vitreous sections to analyze the recently described bacterial cytoplasmic structure, the Stack
    [Feb 2015]

    Publication date: Available online 21 January 2015
    Source:Journal of Structural Biology

    Author(s): Lidia Delgado , Gema Martínez , Carmen López-Iglesias , Elena Mercadé

    Cryo-electron tomography (CET) of plunge-frozen whole bacteria and vitreous sections (CETOVIS) were used to revise and expand the structural knowledge of the “Stack”, a recently described cytoplasmic structure in the Antarctic bacterium Pseudomonas deceptionensis M1T. The advantages of both techniques can be complementarily combined to obtain more reliable insights into cells and their components with three-dimensional imaging at different resolutions. Cryo-electron microscopy (Cryo-EM) and CET of frozen-hydrated P. deceptionensis M1T cells confirmed that Stacks are found at different locations within the cell cytoplasm, in variable number, separately or grouped together, very close to the plasma membrane (PM) and oriented at different angles (from 35° to 90°) to the PM, thus establishing that they were not artifacts of the previous sample preparation methods. CET of plunge-frozen whole bacteria and vitreous sections verified that each Stack consisted of a pile of oval disc-like subunits, each disc being surrounded by a lipid bilayer membrane and separated from each other by a constant distance with a mean value of 5.2±1.3nm. FM4-64 staining and confocal microscopy corroborated the lipid nature of the membrane of the Stacked discs. Stacks did not appear to be invaginations of the PM because no continuity between both membranes was visible when whole bacteria were analyzed. We are still far from deciphering the function of these new structures, but a first experimental attempt links the Stacks with a given phase of the cell replication process.





    Categories: Journal Articles
  • DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly
    [Feb 2015]

    Publication date: Available online 19 January 2015
    Source:Journal of Structural Biology

    Author(s): Andrew D. Hirsh , N.C. Perkins

    While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ 29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA–DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm3) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal.





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

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









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

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









    Categories: Journal Articles
  • Corrigendum to “A clarification of the terms used in comparing semi-automated particle selection algorithms in cryo-EM” [J. Struct. Biol. 175 (2011) 348–352]
    [Feb 2015]

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

    Author(s): Robert Langlois , Joachim Frank







    Categories: Journal Articles
  • Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans
    [Feb 2015]

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

    Author(s): Vera Bin San Chan , Olev Vinn , Chaoyi Li , Xingwen Lu , Anatoliy B. Kudryavtsev , J. William Schopf , Kaimin Shih , Tong Zhang , Vengatesen Thiyagarajan

    The serpulid tubeworm, Hydroides elegans, is an ecologically and economically important species whose biology has been fairly well studied, especially in the context of larval development and settlement on man-made objects (biofouling). Nevertheless, ontogenetic changes associated with calcareous tube composition and structures have not yet been studied. Here, the ultrastructure and composition of the calcareous tubes built by H. elegans was examined in the three early calcifying juvenile stages and in the adult using XRD, FTIR, ICP-OES, SEM and Raman spectroscopy. Ontogenetic shifts in carbonate mineralogy were observed, for example, juvenile tubes contained more amorphous calcium carbonate and were predominantly aragonitic whereas adult tubes were bimineralic with considerably more calcite. The mineral composition gradually shifted during the tube development as shown by a decrease in Sr/Ca and an increase of Mg/Ca ratios with the tubeworm’s age. The inner tube layer contained calcite, whereas the outer layer contained aragonite. Similarly, the tube complexity in terms of ultrastructure was associated with development. The sequential appearance of unoriented ultrastructures followed by oriented ultrastructures may reflect the evolutionary history of serpulid tube biominerals. As aragonitic structures are more susceptible to dissolution under ocean acidification (OA) conditions but are more difficult to be removed by anti-fouling treatments, the early developmental stages of the tubeworms may be vulnerable to OA but act as the important target for biofouling control.





    Categories: Journal Articles
  • Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance
    [Feb 2015]

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

    Author(s): M. Bangera , R. Panigrahi , S.R. Sagurthi , H.S. Savithri , M.R.N. Murthy

    In many organisms “Universal Stress Proteins” (USPs) are induced in response to a variety of environmental stresses. Here we report the structures of two USPs, YnaF and YdaA from Salmonella typhimurium determined at 1.8Å and 2.4Å resolutions, respectively. YnaF consists of a single USP domain and forms a tetrameric organization stabilized by interactions mediated through chloride ions. YdaA is a larger protein consisting of two tandem USP domains. Two protomers of YdaA associate to form a structure similar to the YnaF tetramer. YdaA showed ATPase activity and an ATP binding motif G-2X-G-9X-G(S/T/N) was found in its C-terminal domain. The residues corresponding to this motif were not conserved in YnaF although YnaF could bind ATP. However, unlike YdaA, YnaF did not hydrolyse ATP in vitro. Disruption of interactions mediated through chloride ions by selected mutations converted YnaF into an ATPase. Residues that might be important for ATP hydrolysis could be identified by comparing the active sites of native and mutant structures. Only the C-terminal domain of YdaA appears to be involved in ATP hydrolysis. The structurally similar N-terminal domain was found to bind a zinc ion near the segment equivalent to the phosphate binding loop of the C-terminal domain. Mass spectrometric analysis showed that YdaA might bind a ligand of approximate molecular weight 800daltons. Structural comparisons suggest that the ligand, probably related to an intermediate in lipid A biosynthesis, might bind at a site close to the zinc ion. Therefore, the N-terminal domain of YdaA binds zinc and might play a role in lipid metabolism. Thus, USPs appear to perform several distinct functions such as ATP hydrolysis, altering membrane properties and chloride sensing.





    Categories: Journal Articles
  • Crystal structures for short-chain pentraxin from zebrafish demonstrate a cyclic trimer with new recognition and effector faces
    [Feb 2015]

    Publication date: Available online 13 January 2015
    Source:Journal of Structural Biology

    Author(s): Rong Chen , Jianxun Qi , Hongyu Yuan , Yanan Wu , Wei Hu , Chun Xia

    Short-chain pentraxins (PTXs), including CRP and SAP, are innate pattern recognition receptors that play vital roles in the recognition and elimination of various pathogenic bacteria by triggering the classical complement pathway through C1q. Similar to antibodies, pentraxins can also activate opsonisation and phagocytosis by interacting with Fc receptors (FcRs). Various structural studies on human PTXs have been performed, but there are no reports about the crystal structure of bony fish pentraxins. Here, the crystal structures of zebrafish PTX (Dare-PTX-Ca and Dare-PTX) are presented. Both Dare-PTX-Ca and Dare-PTX are cyclic trimers, which are new forms of crystallised pentraxins. The structures reveal that the ligand-binding pocket (LBP) in the recognition face of Dare-PTX is deep and narrow. Homology modelling shows that LBPs from different Dare-PTX loci differ in shape, reflecting their specific recognition abilities. Furthermore, in comparison with the structure of hCPR, a new C1q binding mode was identified in Dare-PTX. In addition, the FcR-binding sites of hSAP are partially conserved in Dare-PTX. These results will shed light on the understanding of a primitive PTX in bony fish, which evolved approximately 450 million years ago.





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

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1









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

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1









    Categories: Journal Articles
  • The scrunchworm hypothesis: Transitions between A-DNA and B-DNA provide the driving force for genome packaging in double-stranded DNA bacteriophages
    [Feb 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Stephen C. Harvey

    Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy released by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening–lengthening motions of the B→A→B cycle (“scrunching”), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions.





    Categories: Journal Articles
  • Improvement of protein binding sites prediction by selecting amino acid residues’ features
    [Feb 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Georgina Mirceva , Andrea Kulakov

    One of the main focuses of bioinformatics community is the study of the relationship between the structure of the protein molecules and their functions. In the literature, there are various methods that consider different protein-derived information for predicting protein functions. In our research, we focus on predicting the protein binding sites, which could be used to functionally annotate the protein structures. In this paper we consider a set of sixteen amino acid residues’ features, and by applying various feature selection techniques we estimate their significance. Although the number of features in our case is not high, we perform feature selection in order to improve the prediction power and time complexity of the prediction models. The results show that by applying proper feature selection technique, the predictive performance of the classification algorithms is improved, i.e., by considering the most relevant features we induce more accurate models than if we consider the entire set of features. Furthermore, the model complexity, as well as the training and testing times are decreased by performing feature selection. We also compare our approach with several existing methods for protein binding sites prediction. The results demonstrate that the existing methods considered in this research are specific and applicable to the group of proteins for which the model was developed, while our approach is more generic and can be applied to a wider class of proteins.





    Categories: Journal Articles
  • Stability of the βB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry
    [Feb 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Satoko Akashi , Simin D. Maleknia , Kazumi Saikusa , Kevin M. Downard

    Ion mobility mass spectrometry was employed to study the structure of the βB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of βB3 crystallin across residues 115–126 and 152–166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.





    Categories: Journal Articles