Journal of Structural Biology

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  • A first census of collagen interruptions: collagen’s own stutters and stammers
    [Apr 2014]

    Publication date: Available online 4 April 2014
    Source:Journal of Structural Biology

    Author(s): Jordi Bella

    The repetitive Gly-X-Y sequence is the telltale sign of triple helical domains in collagens and collagen-like proteins. Most collagen sequences contain sporadic interruptions of this pattern, which may have functional roles in molecular flexibility, assembly or molecular recognition. However, the structural signatures of the different interruptions are not well defined. Here, a first comprehensive survey of collagen interruptions on collagen sequences from different taxonomic groups is presented. Amino acid preferences at the sites of interruption and the flanking triplets are analysed separately for metazoan and prokaryotic collagens and the concept of commensurateness between interruptions is introduced. Known structural information from model peptides is used to present a common framework for hydrogen bonding topology and variations in superhelical twist for the different types of interruptions. Several collagen interruptions are further classified here as stutters or stammers in analogy to the heptad breaks observed in alpha-helical coiled coils, and the structural consequences of commensurate interruptions in heterotrimeric collagens are briefly discussed. Data presented here will be useful for further investigation on the relation between structure and function of collagen interruptions.





    Categories: Journal Articles
  • Vitrification of Tokuyasu-style immuno-labeled sections for correlative cryo light microscopy and cryo electron tomography
    [Apr 2014]

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

    Author(s): Erik Bos , Leonie Hussaarts , Jan R.T. van Weering , Mark H. Ellisman , Heidi de Wit , Abraham J. Koster

    We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (Vitrification Of Sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.





    Categories: Journal Articles
  • The structure of AAVrh32.33, a Novel Gene Delivery Vector
    [Apr 2014]

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

    Author(s): Kyle Mikals , Hyun-Joo Nam , Kim Van Vliet , Luk H. Vandenberghe , Lauren E. Mays , Robert McKenna , James M. Wilson , Mavis Agbandje-McKenna

    The Adeno-Associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral two-fold axis, three protrusions surrounding the three-fold axis, and a depression surround a cylindrical channel at the five-fold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.





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

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1









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

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1









    Categories: Journal Articles
  • Automated particle picking for low-contrast macromolecules in cryo-electron microscopy
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Robert Langlois , Jesper Pallesen , Jordan T. Ash , Danny Nam Ho , John L. Rubinstein , Joachim Frank

    Cryo-electron microscopy is an increasingly popular tool for studying the structure and dynamics of biological macromolecules at high resolution. A crucial step in automating single-particle reconstruction of a biological sample is the selection of particle images from a micrograph. We present a novel algorithm for selecting particle images in low-contrast conditions; it proves more effective than the human eye on close-to-focus micrographs, yielding improved or comparable resolution in reconstructions of two macromolecular complexes.





    Categories: Journal Articles
  • Single particle analysis integrated with microscopy: A high-throughput approach for reconstructing icosahedral particles
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Xiaodong Yan , Giovanni Cardone , Xing Zhang , Z. Hong Zhou , Timothy S. Baker

    In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.





    Categories: Journal Articles
  • Key structural arrangements at the C-terminus domain of CETP suggest a potential mechanism for lipid-transfer activity
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Victor García-González , Nadia Gutiérrez-Quintanar , Paola Mendoza-Espinosa , Pilar Brocos , Ángel Piñeiro , Jaime Mas-Oliva

    The cholesteryl-ester transfer protein (CETP) promotes cholesteryl-ester and triglyceride transfer between lipoproteins. We evaluated the secondary structure stability of a series of small peptides derived from the C-terminus of CETP in a wide range of pH’s and lipid mixtures, and studied their capability to carry out disorder-to-order secondary structure transitions dependent of lipids. We report that while a mixture of phosphatidylcholine/cholesteryl-esters forms large aggregated particles, the inclusion of a series of CETP carboxy-terminal peptides in a stable α-helix conformation, allows the formation of small homogeneous micelle-like structures. This phenomenon of lipid ordering was directly connected to secondary structural transitions at the C-terminus domain when lysophosphatidic acid and lysophosphatidylcholine lipids were employed. Circular dichroism, cosedimentation experiments, electron microscopy, as well as molecular dynamics simulations confirm this phenomenon. When purified CETP is studied, the same type of phenomenon occurs by promoting the reorganization of lipid from large to smaller particles. Our findings extend the emerging view for a novel mechanism of lipid transfer carried out by CETP, assigning its C-terminus domain the property to accomplish lipid ordering through secondary structure disorder-to-order transitions.





    Categories: Journal Articles
  • Local regularization of tilt projections reduces artifacts in electron tomography
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Mauro Maiorca , Coralie Millet , Eric Hanssen , Brian Abbey , Edmund Kazmierczak , Leann Tilley

    Electron tomography produces very high resolution 3D image volumes useful for investigating the structure and function of cellular components. Unfortunately, unavoidable discontinuities and physical constraints in the acquisition geometry lead to a range of artifacts that can affect the reconstructed image. In particular, highly electron dense regions, such as gold nanoparticles, can hide proximal biological structures and degrade the overall quality of the reconstructed tomograms. In this work we introduce a pre-reconstruction non-conservative non-linear isotropic diffusion (NID) filter that automatically identifies and reduces local irregularities in the tilt projections. We illustrate the improvement in quality obtained using this approach for reconstructed tomograms generated from samples of malaria parasite-infected red blood cells. A quantitative and qualitative evaluation for our approach on both simulated and real data is provided.





    Categories: Journal Articles
  • Structural and functional characterization of enamel pigmentation in shrews
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): M. Dumont , T. Tütken , A. Kostka , M.J. Duarte , S. Borodin

    Pigmented tooth enamel occurs in several vertebrate clades, ranging from mammals to fish. Although an iron compound is associated with this orange to red colored pigmentation, its chemical and structural organization within the enamel is unknown. To determine the nature of the iron compound, we investigated heavily pigmented teeth of the northern short-tailed shrew Blarina brevicauda using combined characterization techniques such as scanning and transmission electron microscopy and synchrotron X-ray diffraction. We found that the pigmentation of the enamel with an iron content of around 8wt% results from a close to amorphous magnetite phase deposited around the nm-sized enamel crystals. Furthermore, the influence of the pigmentation on the enamel hardness was determined by nanoindentation measurements. Finally, the biomechanical function and biological context are discussed in light of the obtained results.





    Categories: Journal Articles
  • Robust membrane detection based on tensor voting for electron tomography
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Antonio Martinez-Sanchez , Inmaculada Garcia , Shoh Asano , Vladan Lucic , Jose-Jesus Fernandez

    Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure.





    Categories: Journal Articles
  • Motif Analyzer for protein 3D structures
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Evgeniy Aksianov

    The topology of the protein structure of all-β- or α/β-class is a special arrangement of β-strands within β-sheets (and α-helices surrounding β-sheets) and the order of them along the polypeptide chain. Structural motifs are a subset of strands and/or helices with widely spread topology. Structural motifs are used for classification of protein structure. Because of an increasing variety of known structures, an automatic tool for motif detection is needed. MotAn is an algorithmic detector of structural motifs in a given 3D protein structure. It detects β-hairpins, β-meanders, β-helices, Greek keys, interlocks, jellyrolls, β-α-β-motifs and β-α-β-helices. MotAn was tested on selected SCOP families and shown to be more sensitive detector than the PTGL and PROMOTIF programs. MotAn is available at http://mouse.belozersky.msu.ru/motan.





    Categories: Journal Articles
  • Solution structure of the cyclic-nucleotide binding homology domain of a KCNH channel
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Qingxin Li , Hui Qi Ng , Ho Sup Yoon , Congbao Kang

    The carboxy-terminal region of the KCNH family of potassium channels contains a cyclic-nucleotide binding homology domain (CNBHD) that is important for channel gating and trafficking. The solution structure of the CNBHD of the KCNH potassium of zebrafish was determined using solution NMR spectroscopy. This domain exists as a monomer under solution conditions and adopts a similar fold to that determined by X-ray crystallography. The CNBHD does not bind cAMP because residue Y740 blocks the entry of cyclic-nucleotide to the binding pocket. Relaxation results show that the CNBHD is rigid except that some residues in the loop between β6 and β7 are flexible. Our results will be useful to understand the gating mechanism of KCNH family members through the CNBHD.





    Categories: Journal Articles
  • Structural diversity of a collagen-binding matrix protein from the byssus of blue mussels upon refolding
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Michael H. Suhre , Thomas Scheibel

    Blue mussels firmly adhere to a variety of different substrates by the byssus, an extracorporal structure consisting of several protein threads. These threads are mainly composed of fibrillar collagens called preCols which are embedded in a proteinaceous matrix. One of the two so far identified matrix proteins is the Proximal Thread Matrix Protein 1 (PTMP1). PTMP1 comprises two von Willebrand factor type A-like domains (A1 and A2) in a special arrangement. Here, we describe the refolding of recombinant PTMP1 from inclusion bodies. PTMP1 refolded into two distinct monomeric isoforms. Both isomers exhibited alternative intramolecular disulfide bonds. One of these isomers is thermodynamically favored and presumably represents the native form of PTMP1, while the other isoform is kinetically favored but is likely non-native. Oligomerization during refolding was influenced by, but not strictly dependent on disulfide formation. The conformational stability of PTMP1 indicates an influence of intramolecular disulfides on the native state, but not on unfolding intermediates. Monomeric PTMP1 exhibited a high thermal stability, dependent on the pH of the surrounding environment. Especially under acidic conditions the disulfide bonds were critically involved in thermal stability.





    Categories: Journal Articles
  • The macular degeneration-linked C1QTNF5 (S163) mutation causes higher-order structural rearrangements
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Xiongying Tu , Krzysztof Palczewski

    The C1q-tumor necrosis factor 5 (C1QTNF5) protein plays a significant role in retinal pigmented epithelium (RPE) cellular adhesion. The C1QTNF5 gene is co-transcribed with the frizzled-related protein (MFRP) gene. A Ser-to-Arg mutation at site 163 (S163R) in C1QTNF5 is known to cause late-onset retinal macular degeneration (L-ORMD). Here we also found that C1QTNF5 monomers can multimerize into a bouquet-like octadecamer. We found that a novel intermolecular hydrogen-bond network of S163 that glues adjacent globular heads of C1QTNF5 together was weakened or abolished by the R163 pathogenic mutation. These findings could underlie the structural basis of this protein’s adhesive function and relate to the pathogenesis of its S163R mutation. Additionally, the fact that C1QTNF5 immobilized to a resin selectively enriched detergent extracted membrane-bound MFRP, further confirmed their interaction, implying functions other than cellular adhesion for C1QTNF5.





    Categories: Journal Articles
  • Coupling between inter-helical hydrogen bonding and water dynamics in a proton transporter
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Coral del Val , Luiza Bondar , Ana-Nicoleta Bondar

    Long-distance proton transfers by proton pumps occurs in discrete steps that may involve the direct participation of protein sidechains and water molecules, and coupling of protonation changes to structural rearrangements of the protein matrix. Here we explore the role of inter-helical hydrogen bonding in long-distance protein conformational coupling and dynamics of internal water molecules. From molecular dynamics simulations of wild type and nine different bacteriorhodopsin mutants we find that both intra- and inter-helical hydrogen bonds are important determinants of the local protein structure, dynamics, and water interactions. Based on molecular dynamics and bioinformatics analyses, we identify an aspartate/threonine inter-helical hydrogen-bonding motif involved in controlling the local conformational dynamics. Perturbation of inter-helical hydrogen bonds can couple to rapid changes in water dynamics.





    Categories: Journal Articles
  • Structure of the pseudokinase domain of BIR2, a regulator of BAK1-mediated immune signaling in Arabidopsis
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Bärbel S. Blaum , Sara Mazzotta , Erik R. Nöldeke , Thierry Halter , Johannes Madlung , Birgit Kemmerling , Thilo Stehle

    The BAK1-interacting receptor-like kinase 2 (BIR2) belongs to the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that mediate development and innate immunity in plants and form a monophyletic gene family with the Drosophila Pelle and human interleukin-1 receptor-associated kinases (IRAK). BIR2 is a negative regulator of BAK1-mediated defense mechanisms and cell death responses, yet key residues that are typically required for kinase activity are not present in the BIR2 kinase domain. We have determined the crystal structure of the BIR2 cytosolic domain and show that its nucleotide binding site is occluded. NMR spectroscopy confirmed that neither wild type nor phosphorylation-mimicking mutants of BIR2 bind ATP-analogues in solution, suggesting that BIR2 is a genuine enzymatically inactive pseudokinase. BIR2 is, however, phosphorylated by its target of regulation, BAK1. Using nano LC–MS/MS analysis for site-specific analysis of phosphorylation, we found a high density of BAK1-transphosphorylation sites in the BIR2 juxta membrane domain, a region previously implicated in regulation of RLKs. Our findings provide a structural basis to better understand signaling through kinase-dead domains that are predicted to account for 20% of all Arabidopsis RLKs and 10% of all human kinases.





    Categories: Journal Articles
  • Web server for tilt-pair validation of single particle maps from electron cryomicroscopy
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Sebastian Wasilewski , Peter B. Rosenthal

    Three-dimensional structures of biological assemblies may be calculated from images of single particles obtained by electron cryomicroscopy. A key step is the correct determination of the orientation of the particle in individual image projections. A useful tool for validation of the quality of a 3D map and its consistency with images is tilt-pair analysis. In a successful tilt-pair test, the relative angle between orientations assigned to each image of a tilt-pair agrees with the known relative rotation angle of the microscope specimen holder during the experiment. To make the procedure easy to apply to the increasing number of single particle maps, we have developed software and a web server for tilt-pair analysis. The tilt-pair analysis program reports the overall agreement of the assigned orientations with the known tilt angle and axis of the experiment and the distribution of tilt transformations for individual particles recorded in a single image field. We illustrate application of the validation tool to several single particle specimens and describe how to interpret the scores.





    Categories: Journal Articles
  • Structural dynamics of V3 loop in a trimeric ambiance, a molecular dynamics study on gp120–CD4 trimeric mimic
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Balasubramanian Chandramouli , Giovanni Chillemi , Alessandro Desideri

    Entry of HIV virus into the host cell is initiated by the interaction of its surface exposed gp120 protein with the cell surface CD4 receptor and a co-receptor that can be either CCR5 or CXCR4. The third variable region (V3 loop) of gp120 has an important role in co-receptor selection by gp120 and forms an epitope for neutralizing antibodies. In this work the dynamical behavior of the V3 loop in a trimeric environment has been investigated by generating an atomistic trimer model of gp120–CD4 complex and has been compared with the result of a monomeric gp120–CD4 complex. The main results coming from this work are that the three V3 loops belonging to the three subunits of the trimer display a different dynamical behavior in terms of its flexibility, spatial orientation, motion along the principal modes, conformations, solvent exposure and electrostatic potential distribution. We propose that the ability of the V3 loop to present, in the trimeric environment, simultaneous multiple alternative conformations that increase its capability of co-receptor recognition, is at least in part due to the effect of electrostatic potential generated by two subunits over the third one.





    Categories: Journal Articles
  • A simple Fourier filter for suppression of the missing wedge ray artefacts in single-axis electron tomographic reconstructions
    [Apr 2014]

    Publication date: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    Author(s): Lubomír Kováčik , Sami Kerïeche , Johanna L. Höög , Pavel Jůda , Pavel Matula , Ivan Raška

    The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable – the missing wedge. Since this region is sharply delimited from the area of available data, the reconstructed signal is typically hampered by convolution with its impulse response, which gives rise to the well-known missing wedge artefacts in 3D reconstructions. Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology. Therefore we have designed a simple Fourier angular filter that effectively suppresses the ray artefacts in the single-axis tilting projection acquisition scheme, making single-axis tomographic reconstructions easier to interpret in particular at low signal-to-noise ratio in acquired projections. The proposed filter can be easily incorporated into current electron tomographic reconstruction schemes.





    Categories: Journal Articles