Journal of Structural Biology

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

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

    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
  • A marker-free automatic alignment method based on scale-invariant features
    [Apr 2014]

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

    Author(s): Renmin Han , Fa Zhang , Xiaohua Wan , Jose-Jesus Fernández , Fei Sun , Zhiyong Liu

    In electron tomography, alignment accuracy is critical for high-resolution reconstruction. However, the automatic alignment of a tilt series without fiducial markers remains a challenge. Here, we propose a new alignment method based on Scale-Invariant Feature Transform (SIFT) for marker-free alignment. The method covers the detection and localization of interest points (features), feature matching, feature tracking and optimization of projection parameters. The proposed method implements a highly reliable matching strategy and tracking model to detect a huge number of feature tracks. Furthermore, an incremental bundle adjustment method is devised to tolerate noise data and ensure the accurate estimation of projection parameters. Our method was evaluated with a number of experimental data, and the results exhibit an improved alignment accuracy comparable with current fiducial marker alignment and subsequent higher resolution of tomography.





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

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

    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
  • The structure of the N-terminal domain of the Legionella protein SidC
    [Apr 2014]

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

    Author(s): Emerich Mihai Gazdag , Stefan Schöbel , Alexander V. Shkumatov , Roger S. Goody , Aymelt Itzen

    The Gram-negative bacterium Legionella pneumophila is the causative agent of Legionnaires’ disease. During infection of eukaryotic cells, the bacterium releases about 300 different bacterial effector molecules that aid in the establishment of the Legionella-containing vacuole (LCV) among which SidC is one of these secreted proteins. However, apart from membrane lipid binding the function of SidC remains elusive. In order to characterize SidC further, we have determined the crystal structure of the N-terminal domain of SidC (amino acids 1–609, referred to as SidC-N) at 2.4Å resolution. SidC-N reveals a novel fold in which 4 potential subdomains (A–D) are arranged in a crescent-like structure. None of these subdomains currently has any known structural homologues, raising the question of how this fold has evolved. These domains are highly interconnected, with a low degree of flexibility towards each other. Due to the extended arrangement of the subdomains, SidC-N may contain multiple binding sites for potential interaction partners.





    Categories: Journal Articles
  • 3D domain swapping in a chimeric c-Src SH3 domain takes place through two hinge loops
    [Apr 2014]

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

    Author(s): Ana Cámara-Artigas , Sergio Martínez-Rodríguez , Emilia Ortiz-Salmerón , José M. Martín-García

    In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.





    Categories: Journal Articles
  • 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: April 2014
    Source:Journal of Structural Biology, Volume 186, Issue 1

    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
  • Correlative Light- and Electron Microscopy with chemical tags
    [Apr 2014]

    Publication date: Available online 31 March 2014
    Source:Journal of Structural Biology

    Author(s): Mario Perkovic , Michael Kunz , Ulrike Endesfelder , Stefanie Bunse , Christoph Wigge , Zhou Yu , Victor-Valentin Hodirnau , Margot P. Scheffer , Anja Seybert , Sebastian Malkusch , Erin M. Schuman , Mike Heilemann , Achilleas S. Frangakis

    Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.





    Categories: Journal Articles
  • Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs
    [Apr 2014]

    Publication date: Available online 30 March 2014
    Source:Journal of Structural Biology

    Author(s): Maxim Shatsky , Pablo Arbelaez , Bong-Gyoon Han , Dieter Typke , Steven E. Brenner , Jitendra Malik , Robert M. Glaeser

    Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the “free-hand” procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.





    Categories: Journal Articles
  • 2dx_automator: Implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline
    [Apr 2014]

    Publication date: Available online 28 March 2014
    Source:Journal of Structural Biology

    Author(s): Sebastian Scherer , Julia Kowal , Mohamed Chami , Venkata Dandey , Marcel Arheit , Philippe Ringler , Henning Stahlberg

    The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein Mlok1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.





    Categories: Journal Articles
  • A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein
    [Apr 2014]

    Publication date: Available online 28 March 2014
    Source:Journal of Structural Biology

    Author(s): Emma S. Gleave , Helgo Schmidt , Andrew P. Carter

    Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our S accharomyces cerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.





    Categories: Journal Articles