Journal of Structural Biology

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  • Analysis of acute brain slices by electron microscopy: A correlative light–electron microscopy workflow based on Tokuyasu cryo-sectioning
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Celine Loussert Fonta , Andrew Leis , Cliff Mathisen , David S. Bouvier , Willy Blanchard , Andrea Volterra , Ben Lich , Bruno M. Humbel

    Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu’s protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.





    Categories: Journal Articles
  • The structure of ibuprofen bound to cyclooxygenase-2
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Benjamin J. Orlando , Michael J. Lucido , Michael G. Malkowski

    The cyclooxygenases (COX-1 and COX-2) catalyze the rate-limiting step in the biosynthesis of prostaglandins, and are the pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). Ibuprofen (IBP) is one of the most commonly available over-the-counter pharmaceuticals in the world. The anti-inflammatory and analgesic properties of IBP are thought to arise from inhibition of COX-2 rather than COX-1. While an X-ray crystal structure of IBP bound to COX-1 has been solved, no such structure exists for the cognate isoform COX-2. We have determined the crystal structure of muCOX-2 with a racemic mixture of (R/S)-IBP. Our structure reveals that only the S-isomer of IBP was bound, indicating that the S-isomer possesses higher affinity for COX-2 than the R-isomer. Mutational analysis of Arg-120 and Tyr-355 at the entrance of the cyclooxygenase channel confirmed their role in binding and inhibition of COX-2 by IBP. Our results provide the first atomic level detail of the interaction between IBP and COX-2.





    Categories: Journal Articles
  • Structure of the c-Src-SH3 domain in complex with a proline-rich motif of NS5A protein from the hepatitis C virus
    [Jan 2015]

    Publication date: January 2015
    Source:Journal of Structural Biology, Volume 189, Issue 1

    Author(s): Julio Bacarizo , Sergio Martínez-Rodríguez , Ana Cámara-Artigas

    The non-structural hepatitis C virus proteins NS5A and NS5B form a complex through interaction with the SH2 and SH3 domains of the non-receptor Src tyrosine kinase, which seems essential for viral replication. We have crystallized the complex between the SH3 domain of the c-Src tyrosine kinase and the C-terminal proline rich motif of the NS5A protein (A349PPIPPPRRKR359). Crystals obtained at neutral pH belong to the space group I41, with a single molecule of the SH3/NS5A complex at the asymmetric unit. The NS5A peptide is bound in a reverse orientation (class II) and the comparison of this structure with those of the high affinity synthetic peptides APP12 and VSL12 shows some important differences at the salt bridge that drives the peptide orientation. Further conformational changes in residues placed apart from the binding site also seem to play an important role in the binding orientation of this peptide. Our results show the interaction of the SH3 domain of the c-Src tyrosine kinase with a proline rich motif in the NS5A protein and point to their potential interaction in vivo.





    Categories: Journal Articles
  • Large area sub-micron chemical imaging of magnesium in sea urchin teeth
    [Jan 2015]

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

    Author(s): Admir Masic , James C. Weaver

    The heterogeneous and site-specific incorporation of inorganic ions can profoundly influence the local mechanical properties of damage tolerant biological composites. Using the sea urchin tooth as a research model, we describe a multi-technique approach to spatially map the distribution of magnesium in this complex multiphase system. Through the combined use of 16-bit backscattered scanning electron microscopy, multi-channel energy dispersive spectroscopy elemental mapping, and diffraction-limited confocal Raman spectroscopy, we demonstrate a new set of high throughput, multi-spectral, high resolution methods for the large scale characterization of mineralized biological materials. In addition, instrument hardware and data collection protocols can be modified such that several of these measurements can be performed on irregularly shaped samples with complex surface geometries and without the need for extensive sample preparation. Using these approaches, in conjunction with whole animal micro-computed tomography studies, we have been able to spatially resolve micron and sub-micron structural features across macroscopic length scales on entire urchin tooth cross-sections and correlate these complex morphological features with local variability in elemental composition.





    Categories: Journal Articles
  • An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles
    [Jan 2015]

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

    Author(s): Richard McGonigle , Wei Boon Yap , Swee Tin Ong , Derek Gatherer , Saskia E. Bakker , Wen Siang Tan , David Bhella

    Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.





    Categories: Journal Articles
  • Use of a “silver bullet” to resolve crystal lattice dislocation disorder: A cobalamin complex of Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis
    [Jan 2015]

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

    Author(s): Thomas Lagautriere , Ghader Bashiri , Edward N. Baker

    The use of small molecules as “silver bullets” that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order–disorder phenomenon that complicated the structural analysis of the enzyme Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order–disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.





    Categories: Journal Articles
  • Seeing tobacco mosaic virus through direct electron detectors
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Simon A. Fromm , Tanmay A.M. Bharat , Arjen J. Jakobi , Wim J.H. Hagen , Carsten Sachse

    With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.





    Categories: Journal Articles
  • Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Jose-Ignacio Agulleiro , Jose-Jesus Fernandez

    Tomo3D is a program for fast tomographic reconstruction on multicore computers. Its high speed stems from code optimization, vectorization with Streaming SIMD Extensions (SSE), multithreading and optimization of disk access. Recently, Advanced Vector eXtensions (AVX) have been introduced in the x86 processor architecture. Compared to SSE, AVX double the number of simultaneous operations, thus pointing to a potential twofold gain in speed. However, in practice, achieving this potential is extremely difficult. Here, we provide a technical description and an assessment of the optimizations included in Tomo3D to take advantage of AVX instructions. Tomo3D 2.0 allows huge reconstructions to be calculated in standard computers in a matter of minutes. Thus, it will be a valuable tool for electron tomography studies with increasing resolution needs.





    Categories: Journal Articles
  • On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF
    [Jan 2015]

    Publication date: Available online 9 December 2014
    Source:Journal of Structural Biology

    Author(s): Marcia Folly-Klan , Bastien Sancerne , Eric Alix , Craig R. Roy , Jacqueline Cherfils , Valérie Campanacci

    A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1Å with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.





    Categories: Journal Articles
  • Semi-automated selection of cryo-EM particles in RELION-1.3
    [Jan 2015]

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

    Author(s): Sjors H.W. Scheres

    The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.





    Categories: Journal Articles
  • Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: The example of BV-2 microglia cells
    [Jan 2015]

    Publication date: Available online 5 December 2014
    Source:Journal of Structural Biology

    Author(s): E. Gavgiotaki , G. Filippidis , M. Kalognomou , A.A. Tsouko , I. Skordos , C. Fotakis , I. Athanassakis

    Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.





    Categories: Journal Articles
  • Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization
    [Jan 2015]

    Publication date: Available online 3 December 2014
    Source:Journal of Structural Biology

    Author(s): R. Lasala , N. Coudray , A. Abdine , Z. Zhang , M. Lopez-Redondo , R. Kirshenbaum , J. Alexopoulos , Z. Zolnai , D.L. Stokes , I. Ubarretxena-Belandia

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles
  • Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Yingying Zheng , Tzu-Ping Ko , Hong Sun , Chun-Hsiang Huang , Jianjun Pei , Riyong Qiu , Andrew H.-J. Wang , Juergen Wiegel , Weilan Shao , Rey-Ting Guo

    The Rex-family repressors sense redox levels by alternative binding to NADH or NAD+. Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200T. It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD+/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD+, give rise to two sets of protein–ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.





    Categories: Journal Articles
  • Biogenic nanospheres of amorphous carbonated Ca–Mg phosphate within the periostracum of the green mussel Perna viridis
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Jun Xu , Gangsheng Zhang

    Recently there is increasing evidence that the shell biomineralization proceeds via an amorphous precursor route. Therefore, the search for and investigation of amorphous biominerals in bivalve shells are of great importance and interest. Here, using a scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), and Fourier transform infrared spectrometer (FTIR), we investigate the microstructure and mineralogy of the periostracum in Perna viridis. We find that: (1) the periostracum has three layers, of which the inner and outer layer are of proteins, while the middle layer is mineralized with nanospheres of amorphous biominerals; (2) the nanospheres are of amorphous carbonated Ca–Mg phosphate (ACCP), where the CO3 2 −/PO4 3 − weight ratio is estimated to be ∼0.3, and the Ca/P and Ca/Mg atomic ratio is ∼1.4 and 1.6, respectively; (3) the nanospheres, with a diameter of 43–106nm, are found to assemble into spherules with a diameter of 160–500nm, which are further organized into parallel microlayers separated by the proteins; and (4) the nanospheres are assumed to function as the pH stabilizer to facilitate the shell’s initial mineralization. Finally, we expect that these findings will advance our understanding of the shell’s biomineralization process.





    Categories: Journal Articles
  • Amino acid sequence homologies in the hard keratins of birds and reptiles, and their implications for molecular structure and physical properties
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): R.D. Bruce Fraser , David A.D. Parry

    Avian and reptilian epidermal appendages such as feathers, claws and scales exhibit a filament–matrix texture. Previous studies have established that both components reside within the same single-chain molecule. In the present study the homology in a wide range of aligned sequences is used to gain insights into the structure and function of the molecular segments associated with the filament and with the matrix. The notion that all molecules contain a β-rich 34-residue segment associated with the framework of the filament is reinforced by the present study. In addition, the residues involved in the polymerization of the molecules to form filaments are identified. In the Archosaurs (birds, crocodiles and turtles), and the Squamates (snakes and lizards) segments rich in glycine and tyrosine can be identified in the C-terminal domain. In Rhynocephalians (tuataras) and Squamates a similar segment is inserted at a specific point in the N-terminal domain. In some Archosaurian appendages (both avian and reptilian) segments rich in charged residues and cysteine are found in the N-terminal domain. The likely effect of these segments will be to soften the tissue without compromising its insolubility. The structure and role of the various molecular segments identified in this study and the way in which they might manifest themselves in terms of the physical properties of the particular epidermal appendage in which they appear are also discussed.





    Categories: Journal Articles
  • Thalidomide mimics uridine binding to an aromatic cage in cereblon
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Marcus D. Hartmann , Iuliia Boichenko , Murray Coles , Fabio Zanini , Andrei N. Lupas , Birte Hernandez Alvarez

    Thalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.





    Categories: Journal Articles
  • Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Xiaojian Gong , Xiaofang Chen , Dongmin Yu , Nannan Zhang , Zhongliang Zhu , Liwen Niu , Yuxin Mao , Honghua Ge

    Dephospho-CoA kinase (DPCK; EC 2.7.1.24) catalyzes the final step in the coenzyme A biosynthetic pathway. DPCK transfers a phosphate group from ATP to the 3-hydroxyl group of the ribose of dephosphocoenzyme A (dCoA) to yield CoA and ADP. Upon the binding of ligands, large conformational changes is induced in DPCKs, as well as in many other kinases, to shield the bound ATP in their catalytic site from the futile hydrolysis by bulk water molecules. To investigate the molecular mechanisms underlying the phosphoryl transfer during DPCK catalytic cycle, we determined the crystal structures of the Legionella pneumophila DPCK (LpDPCK) both in its apo-form and in complex with ATP. The structures reveal that LpDPCK comprises of three domains, the classical core domain, the CoA domain, and the LID domain, which are packed together to create a central cavity for substrate-binding and enzymatic catalysis. The binding of ATP induces large conformational changes, including a hinge-bending motion of the CoA binding domain and the “helix to loop” conformational change of the P-loop. Finally, modeling of a dCoA molecule to the enzyme provides insights into the catalytic mechanism of DPCK.





    Categories: Journal Articles
  • Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Henry P. Schwarcz , Elizabeth A. McNally , Gianluigi A. Botton

    In a previous study we showed that most of the mineral in bone is present in the form of “mineral structures”, 5–6nm-thick, elongated plates which surround and are oriented parallel to collagen fibrils. Using dark-field transmission electron microscopy, we viewed mineral structures in ion-milled sections of cortical human bone cut parallel to the collagen fibrils. Within the mineral structures we observe single crystals of apatite averaging 5.8±2.7nm in width and 28±19nm in length, their long axes oriented parallel to the fibril axis. Some appear to be composite, co-aligned crystals as thin as 2nm. From their similarity to TEM images of crystals liberated from deproteinated bone we infer that we are viewing sections through platy crystals of apatite that are assembled together to form the mineral structures.





    Categories: Journal Articles