Journal of Structural Biology

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

    Publication date: May 2014
    Source:Journal of Structural Biology, Volume 186, Issue 2

    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
  • Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: Towards their structure stability and homogeneity
    [Jun 2014]

    Publication date: May 2014
    Source:Journal of Structural Biology, Volume 186, Issue 2

    Author(s): Susana Galante-Oliveira , Raquel Marçal , Fernanda Guimarães , Jorge Soares , José Carlos Lopes , Jorge Machado , Carlos Barroso

    Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures’ mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates – aragonite and calcite – coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000cm−1 consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.





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

    Publication date: May 2014
    Source:Journal of Structural Biology, Volume 186, Issue 2

    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
  • The structure of AAVrh32.33, a novel gene delivery vector
    [Jun 2014]

    Publication date: May 2014
    Source:Journal of Structural Biology, Volume 186, Issue 2

    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 3.5Å 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 twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold 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
  • Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies
    [Jun 2014]

    Publication date: Available online 26 April 2014
    Source:Journal of Structural Biology

    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.





    Categories: Journal Articles
  • Molecular dynamics investigation of the active site dynamics of mycobacterial cyclopropane synthase during various stages of the cyclopropanation process
    [Jun 2014]

    Publication date: Available online 26 April 2014
    Source:Journal of Structural Biology

    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.





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

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









    Categories: Journal Articles
  • Table of Contents / barcode
    [Jun 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
    [Jun 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
    [Jun 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
  • Cover 2 - Editorial Board
    [Jun 2014]

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









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

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









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

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

    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
  • Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor
    [Jun 2014]

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

    Author(s): Hedda U. Ferris , Kornelius Zeth , Michael Hulko , Stanislaw Dunin-Horkawicz , Andrei N. Lupas

    Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15–20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation–demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.





    Categories: Journal Articles
  • A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity
    [Jun 2014]

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

    Author(s): Marcus D. Hartmann , Stanislaw Dunin-Horkawicz , Michael Hulko , Jörg Martin , Murray Coles , Andrei N. Lupas

    Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains.





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

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

    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
  • Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation
    [Jun 2014]

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

    Author(s): Hedda U. Ferris , Murray Coles , Andrei N. Lupas , Marcus D. Hartmann

    Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.





    Categories: Journal Articles
  • Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors
    [Jun 2014]

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

    Author(s): Silvia Deiss , Birte Hernandez Alvarez , Kerstin Bär , Carolin P. Ewers , Murray Coles , Reinhard Albrecht , Marcus D. Hartmann

    This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date.





    Categories: Journal Articles