Journal of Structural Biology

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  • Corrigendum to “Structure of MST2 SARAH domain provides insights into its interaction with RAPL” [J. Struct. Biol. 185 (2014) 366–374]
    [Apr 2014]

    Publication date: Available online 15 March 2014
    Source:Journal of Structural Biology

    Author(s): Guoguang Liu , Zhubing Shi , Shi Jiao , Zhenzhen Zhang , Wenjia Wang , Cuicui Chen , Qian Hao , Meng Zhang , Miao Feng , Liang Xu , Zhen Zhang , Zhaocai Zhou , Min Zhang







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

    Publication date: Available online 14 March 2014
    Source:Journal of Structural Biology

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

    Publication date: Available online 14 March 2014
    Source:Journal of Structural Biology

    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
  • Three-dimensional structure of minipig fibrolamellar bone: Adaptation to axial loading
    [Apr 2014]

    Publication date: Available online 14 March 2014
    Source:Journal of Structural Biology

    Author(s): Rotem Almany Magal , Natalie Reznikov , Ron Shahar , Steve Weiner

    Fibrolamellar bone is transiently produced by large, fast growing mammals. The fibrolamellar bone unit is initially formed by elaboration of a network of blood vessels. This is followed by the deposition of a thin, porous and hypercalcified layer, then by the infilling of the vascular cavities by the sequential deposition of a relatively thick rapidly forming bone on both sides of the hypercalcified layer, and finally by lamellar bone. We investigated the 3D structure of the collagenous network of fibrolamellar bone from the femora of a young minipig using mainly the FIB–SEM dual beam microscope and the Serial Surface View method. This enabled us to identify the fibril orientation, the canalicular network organization and other structural motifs within each element of the fibrolamellar unit. The first formed primary hypercalcified layer (PHL) is composed of fibril arrays and multiple small pores, and appears to have an isotropic structure. The major bone component is deposited on both sides of the PHL, and is composed of collagen fibrils with a preferred orientation, mainly aligned parallel to the bone long axis. This bone component is therefore parallel-fibered bone and not woven bone. We also observed that the collagen fibers are organized into bundles. The lamellar bone has most of its collagen fibrils aligned with the bone long axis. This study therefore shows that the large majority of collagen fibrils in fibrolamellar bone are aligned with the bone long axis. This anisotropic structure therefore appears to be adapted to loading along the bone long axis.





    Categories: Journal Articles
  • Two and three dimensional characterization of Zucchini Yellow Mosaic Virus induced structural alterations in Cucurbita pepo L. plants
    [Apr 2014]

    Publication date: Available online 13 March 2014
    Source:Journal of Structural Biology

    Author(s): Günther Zellnig , Michael Herbert Pöckl , Stefan Möstl , Bernd Zechmann

    Infection of plants by Zucchini Yellow Mosaic Virus (ZYMV) induces severe ultrastructural changes. The aim of this study was to investigate ultrastructural changes during ZYMV-infection in Cucurbita pepo L. plants on the two and three dimensional (2D and 3D) level and to correlate these changes with the spread of ZYMV throughout the plant by transmission electron microscopy (TEM) and image analysis. This study revealed that after inoculation of the cotyledons ZYMV moved into roots [3days post inoculation (dpi)], then moved upwards into the stem and apical meristem (5dpi), then into the first true leaf (7dpi) and could finally be found in all plant parts (9dpi). ZYMV-infected cells contained viral inclusion bodies in the form of cylindrical inclusions (CIs). These CIs occurred in four different forms throughout the cytosol of roots and leaves: scrolls and pinwheels when cut transversely and long tubular structures and bundles of filaments when cut longitudinally. 3D reconstruction of ZYMV-infected cells containing scrolls revealed that they form long tubes throughout the cytosol. The majority has a preferred orientation and an average length and width of 3μm and 120nm, respectively. Image analysis revealed an increased size of cells and vacuoles (107% and 447%, respectively) in younger ZYMV-infected leaves leading to a similar ratio of cytoplasm to vacuole (about 1:1) in older and younger ZYMV-infected leaves which indicates advanced cell growth in younger tissues. The collected data advances the current knowledge about ZYMV-induced ultrastructural changes in Cucurbita pepo.





    Categories: Journal Articles
  • Effect of sequence features on assembly of spider silk block copolymers
    [Apr 2014]

    Publication date: Available online 12 March 2014
    Source:Journal of Structural Biology

    Author(s): Olena S. Tokareva , Shangchao Lin , Matthew M. Jacobsen , Wenwen Huang , Daniel Rizzo , David Li , Marc Simon , Cristian Staii , Peggy Cebe , Joyce Y. Wong , Markus J. Buehler , David L. Kaplan

    Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.





    Categories: Journal Articles
  • Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system
    [Apr 2014]

    Publication date: Available online 12 March 2014
    Source:Journal of Structural Biology

    Author(s): Tim Kükenshöner , Daniel Wohlwend , Christoph Niemöller , Padmarupa Dondapati , Janina Speck , Adebola V. Adeniran , Anita Nieth , Stefan Gerhardt , Oliver Einsle , Kristian M. Müller , Katja M. Arndt

    The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.





    Categories: Journal Articles
  • Rotationally invariant image representation for viewing direction classification in cryo-EM
    [Apr 2014]

    Publication date: Available online 12 March 2014
    Source:Journal of Structural Biology

    Author(s): Zhizhen Zhao , Amit Singer

    We introduce a new rotationally invariant viewing angle classification method for identifying, among a large number of cryo-EM projection images, similar views without prior knowledge of the molecule. Our rotationally invariant features are based on the bispectrum. Each image is denoised and compressed using steerable principal component analysis (PCA) such that rotating an image is equivalent to phase shifting the expansion coefficients. Thus we are able to extend the theory of bispectrum of 1D periodic signals to 2D images. The randomized PCA algorithm is then used to efficiently reduce the dimensionality of the bispectrum coefficients, enabling fast computation of the similarity between any pair of images. The nearest neighbors provide an initial classification of similar viewing angles. In this way, rotational alignment is only performed for images with their nearest neighbors. The initial nearest neighbor classification and alignment are further improved by a new classification method called vector diffusion maps. Our pipeline for viewing angle classification and alignment is experimentally shown to be faster and more accurate than reference-free alignment with rotationally invariant K-means clustering, MSA/MRA 2D classification, and their modern approximations.





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

    Publication date: Available online 10 March 2014
    Source:Journal of Structural Biology

    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
  • Crystal structure of the extracellular juxtamembrane region of Robo1
    [Apr 2014]

    Publication date: Available online 6 March 2014
    Source:Journal of Structural Biology

    Author(s): Reut Barak , Roxane Lahmi , Lada Gevorkyan-Airapetov , Eliad Levy , Amit Tzur , Yarden Opatowsky

    Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.





    Categories: Journal Articles
  • Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium
    [Apr 2014]

    Publication date: Available online 6 March 2014
    Source:Journal of Structural Biology

    Author(s): Cynthia M. Rodenburg , Sylvia A. McPherson , Charles L. Turnbough Jr. , Terje Dokland

    Bacillus anthracis and other pathogenic Bacillus species form spores that are surrounded by an exosporium, a balloon-like layer that acts as the outer permeability barrier of the spore and contributes to spore survival and virulence. The exosporium consists of a hair-like nap and a paracrystalline basal layer. The filaments of the nap are comprised of trimers of the collagen-like glycoprotein BclA, while the basal layer contains approximately 20 different proteins. One of these proteins, BxpB, forms tight complexes with BclA and is required for attachment of essentially all BclA filaments to the basal layer. Another basal layer protein, ExsB, is required for the stable attachment of the exosporium to the spore. To determine the organization of BclA and BxpB within the exosporium, we used cryo-electron microscopy, cryo-sectioning and crystallographic analysis of negatively stained exosporium fragments to compare wildtype spores and mutant spores lacking BclA, BxpB or ExsB (ΔbclA, ΔbxpB and ΔexsB spores, respectively). The trimeric BclA filaments are attached to basal layer surface protrusions that appear to be trimers of BxpB. The protrusions interact with a crystalline layer of hexagonal subunits formed by other basal layer proteins. Although ΔbxpB spores retain the hexagonal subunits, the basal layer is not organized with crystalline order and lacks basal layer protrusions and most BclA filaments, indicating a central role for BxpB in exosporium organization.





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

    Publication date: Available online 6 March 2014
    Source:Journal of Structural Biology

    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: Available online 5 March 2014
    Source:Journal of Structural Biology

    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
  • Motif Analyzer for protein 3D structures
    [Apr 2014]

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

    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
  • Cover 2 - Editorial Board
    [Apr 2014]

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3









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

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3









    Categories: Journal Articles
  • Super-resolution imaging of Escherichia coli nucleoids reveals highly structured and asymmetric segregation during fast growth
    [Apr 2014]

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3

    Author(s): Christoph Spahn , Ulrike Endesfelder , Mike Heilemann

    Bacterial replication and chromosome segregation are highly organized both in space and in time. However, spatial analysis is hampered by the resolution limit of conventional fluorescence microscopy. In this study, we incubate rapidly-growing Escherichia coli with 5-ethynyl-2′-deoxyuridine (EdU), label the resulting EdU-DNA with photoswitchable fluorophores, and image incorporated molecules with an average experimental precision of 13nm. During the segregation process, nucleoids develop highly-defined and cell-cycle dependent hetero-structures, which contain discrete DNA fibers with diameters far below the diffraction limit. Strikingly, these structures appear temporally shifted between sister chromosomes, an asymmetry which accumulates for ongoing replication rounds. Moreover, nucleoid positioning and expansion along the bacterial length axis fit into an elongation-mediated segregation model in fast growing E. coli cultures. This is supported by close proximity of the nucleoids to the bacterial plasma membrane, the nature of the observed hetero-structures and recently found interactions of membrane-associated proteins with DNA.





    Categories: Journal Articles
  • Exploiting radiation damage to map proteins in nucleoprotein complexes: The internal structure of bacteriophage T7
    [Apr 2014]

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3

    Author(s): Naiqian Cheng , Weimin Wu , Norman R. Watts , Alasdair C. Steven

    In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique (“bubblegram imaging”) by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10s) intervals between exposures of 17electrons/Å2 each, bubbling starts in the third exposure, with 1–4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.





    Categories: Journal Articles
  • Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid
    [Apr 2014]

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3

    Author(s): Ken Okada , Clement Angkawidjaja , Yuichi Koga , Shigenori Kanaya

    Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff’s base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms.





    Categories: Journal Articles
  • Single particle 3D reconstruction for 2D crystal images of membrane proteins
    [Apr 2014]

    Publication date: March 2014
    Source:Journal of Structural Biology, Volume 185, Issue 3

    Author(s): Sebastian Scherer , Marcel Arheit , Julia Kowal , Xiangyan Zeng , Henning Stahlberg

    In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images.





    Categories: Journal Articles