Journal of Structural Biology

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  • Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy
    [Aug 2014]

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

    Author(s): Jason Z. Cui , Arash Y. Tehrani , Kimberly A. Jett , Pascal Bernatchez , Cornelis van Breemen , Mitra Esfandiarei

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.





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  • Iron-rich ferritin in the hypoxia-tolerant rodent Spalax ehrenbergi: A naturally-occurring biomarker confirms the internalization and pathways of intracellular macromolecules
    [Aug 2014]

    Publication date: Available online 19 July 2014
    Source:Journal of Structural Biology

    Author(s): Theodore C. Iancu , Talmon Arad , Imad Shams , Irena Manov

    The discovery of pits/caveolae in the plasmalemma advanced the study of macromolecule internalization. “Transcytosis” describes the transport of macromolecular cargo from one front of a polarized cell to the other within membrane-bounded carrier(s), via endocytosis, intracellular trafficking and exocytosis. Clathrin-mediated transcytosis is used extensively by epithelial cells, while caveolae-mediated transcytosis mostly occurs in endothelial cells. The internalization pathways were monitored by various markers, including radioisotopes, nanoparticles, enzymes, immunostains, and fluorophores. We describe an internalization pathway identified using a naturally-occurring biomarker, in vivo assembled ferritin, containing electron-dense iron cores. Iron, an essential trace metal for most living species and iron homeostasis, is crucial for cellular life. Ferritin is a ubiquitous and highly conserved archeoprotein whose main function is to store a reserve iron supply inside the cytoplasm in a non-toxic form. Ferritin is present in all organisms which have a metabolic requirement for iron and in even in organisms whose taxonomic rank is very low. The newborns of the blind mole, Spalax ehrenbergi, are born and live in a hypoxic environment and have significant iron overload in their liver and heart, but their iron metabolism has not been previously studied. These newborns, which are evolutionarily adapted to fluctuations in the environmental oxygen, have a unique ability to sequester transplacental iron and store it in ferritin without any signs of iron toxicity. Using the ferrihydrite cores of ferritin, we were able to monitor the ferritin internalization from portals of its entry into the cytosol of hepatocytes and cardiomyocytes and into the lysosomes.
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  • 3D Raman mapping of the collagen fibril orientation in human osteonal lamellae
    [Aug 2014]

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

    Author(s): Susanne Schrof , Peter Varga , Leonardo Galvis , Kay Raum , Admir Masic

    Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues.





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  • Cover 2 - Editorial Board
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









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

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1









    Categories: Journal Articles
  • Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Guimei Yu , Frank Vago , Dongsheng Zhang , Jonathan E. Snyder , Rui Yan , Ci Zhang , Christopher Benjamin , Xi Jiang , Richard J. Kuhn , Philip Serwer , David H. Thompson , Wen Jiang

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged E scherichia coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.





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  • M-free: Scoring the reference bias in sub-tomogram averaging and template matching
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Zhou Yu , Achilleas S. Frangakis

    Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.





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  • Crystal structure of the transport unit of the autotransporter adhesin involved in diffuse adherence from Escherichia coli
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Iris Gawarzewski , Frank DiMaio , Elisa Winterer , Britta Tschapek , Sander H.J. Smits , Joachim Jose , Lutz Schmitt

    Several serious gastrointestinal diseases, which are widespread all over the world, are caused by enteropathogenic Escherichia coli. The monomeric autotransporter AIDA-I (adhesin involved in diffuse adherence) represents an important virulence factor of these strains and is involved in adhesion, biofilm formation, aggregation and invasion into host cells. Here, we present the crystal structure of the transport unit of AIDA-I at 3.0Å resolution, which forms a 12-stranded β-barrel harboring the linker domain in its pore. Mutagenesis studies of the C-terminal amino acid demonstrated the great impact of this terminal residue on membrane integration of AIDA-I and passenger translocation.





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  • Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Sandra Hoefgen , Ina Coburger , Dirk Roeser , Yvonne Schaub , Sven O. Dahms , Manuel E. Than

    The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer’s disease (AD). In addition, many physiological functions have been described for APP, including a role in cell–cell- and cell–ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.





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  • Molecular dynamics investigation of the active site dynamics of mycobacterial cyclopropane synthase during various stages of the cyclopropanation process
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Chinmayee Choudhury , U. Deva Priyakumar , G. Narahari Sastry

    Mycobacterial cyclopropane synthase 1 (CmaA1) is one of the most important drug targets in anti tuberculosis drug discovery as it is responsible for cis-cyclopropanation at the distal position of unsaturated mycolates, which is an essential step for the pathogenicity, persistence and drug resistance. Five representative models of CmaA1 which correspond to different stages in the cyclopropanation process have been studied using molecular dynamics (MD) simulations. The MD simulations and structural analyses provide a detailed account of the structural changes in the active sites of CmaA1. CmaA1 has two distinct binding sites, i.e., cofactor binding site (CBS) and acyl substrate binding site (ASBS). The apo state of CmaA1 corresponds to a closed conformation where the CBS is inaccessible due to the existence of H-bond between Pro202 of loop10 (L10) and Asn11 of N-terminal α1 helix. However, cofactor binding leads to the breaking of this H-bond and thus the H-bond is absent in the holo form. The hydrophobic side chains orient towards the inner side of the ASBS upon cofactor binding to create a hydrophobic environment for the substrate. The cofactor and substrate tend to come close to each other facilitated by opening of L10 to exchange the methyl group from the cofactor to the substrate. The MD study also revealed that the system tends to regain the apo conformation within 40ns after releasing the product.





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  • Oxygen–aromatic contacts in intra-strand base pairs: Analysis of high-resolution DNA crystal structures and quantum chemical calculations
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Alok Jain , R.N.V. Krishna Deepak , Ramasubbu Sankararamakrishnan

    Three-dimensional structures of biomolecules are stabilized by a large number of non-covalent interactions and some of them such as van der Waals, electrostatic and hydrogen bond interactions are well characterized. Delocalized π–electron clouds of aromatic residues are known to be involved in cation–π, CH–π, OH–π and π–π interactions. In proteins, many examples have been found in which the backbone carbonyl oxygen of one residue makes close contact with the aromatic center of aromatic residues. Quantum chemical calculations suggest that such contacts may provide stability to the protein secondary structures. In this study, we have systematically analyzed the experimentally determined high-resolution DNA crystal structures and identified 91 examples in which the aromatic center of one base is in close contact (<3.5Ǻ) with the oxygen atom of preceding (Group-I) or succeeding base (Group-II). Examples from Group-I are overwhelmingly observed and cytosine or thymine is the preferred base contributing oxygen atom in Group-I base pairs. A similar analysis of high-resolution RNA structures surprisingly did not yield many examples of oxygen–aromatic contact of similar type between bases. Ab initio quantum chemical calculations on compounds based on DNA crystal structures and model compounds show that interactions between the bases in base pairs with oxygen–aromatic contacts are energetically favorable. Decomposition of interaction energies indicates that dispersion forces are the major cause for energetically stable interaction in these base pairs. We speculate that oxygen–aromatic contacts in intra-strand base pairs in a DNA structure may have biological significance.





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  • In silico analysis and experimental verification of OSR1 kinase – Peptide interaction
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Thomas M. Austin , David P. Nannemann , Samuel L. Deluca , Jens Meiler , Eric Delpire

    The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na+–K+–2Cl− cotransporters (NKCC1–2) and Na+–Cl− cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening.





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  • Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    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
  • CryoEM and image sorting for flexible protein/DNA complexes
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Seth A. Villarreal , Phoebe L. Stewart

    Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.





    Categories: Journal Articles
  • Spherical deconvolution improves quality of single particle reconstruction
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

    Author(s): Gregory P. Kishchenko , Ardean Leith

    One single-particle reconstruction technique is the reconstruction of macromolecules from projection images of randomly oriented particles (SPRR). In SPRR the reliability and consequent interpretation of the final reconstruction is affected by errors arising from incorrect assignment of projection angles to individual particles. In order to improve the resolution of SPRR we studied the influence of imperfect assignment on 3D blurring. We find that this blurring can be described as a Point Spread Function (PSF) that depends on the distance from geometrical center of the reconstructed volume and that blurring is higher at the periphery. This particular PSF can be described by an almost pure tangential angular function with a negligible radial component. We have developed a reliable algorithm for spherical deconvolution of the 3D reconstruction. This spherical deconvolution operation was tested on reconstructions of GroEL and mitochondrial ribosomes. We show that spherical deconvolution improves the quality of SPRR by reducing blurring and enhancing high frequency components, particularly near the periphery of the reconstruction.





    Categories: Journal Articles
  • Corrigendum to “A simple Fourier filter for suppression of the missing wedge ray artefacts in single-axis electron tomographic reconstructions” [J. Struct. Biol. 186(1) (2014) 141–152]
    [Aug 2014]

    Publication date: July 2014
    Source:Journal of Structural Biology, Volume 187, Issue 1

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







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

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3









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

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3









    Categories: Journal Articles
  • Alpbach special issue
    [Aug 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): David A.D. Parry







    Categories: Journal Articles
  • Fifty years of fibrous protein research: A personal retrospective
    [Aug 2014]

    Publication date: June 2014
    Source:Journal of Structural Biology, Volume 186, Issue 3

    Author(s): David A.D. Parry

    As a result of X-ray fiber diffraction studies on fibrous proteins and crystallographic data on fragments derived from them, new experimental techniques across the biophysical and biochemical spectra, sophisticated computer modeling and refinement procedures, widespread use of bioinformatics and improved specimen preparative procedures the structures of many fibrous proteins have now been determined to at least low resolution. In so doing these structures have yielded insight into the relationship that exists between sequence and conformation and this, in turn, has led to improved methodologies for predicting structure from sequence data alone. In this personal retrospective a selection of progress made during the past 50years is discussed in terms of events to which the author has made some contribution.





    Categories: Journal Articles