Journal of Structural Biology

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  • Crystal structure of the effector protein HopA1 from Pseudomonas syringae
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3

    Author(s): Yangshin Park , Inchul Shin , Sangkee Rhee

    Plants have evolved to protect themselves against pathogen attack; in these competitions, many Gram-negative bacteria translocate pathogen-originated proteins known as effectors directly into plant cells to interfere with cellular processes. Effector-triggered immunity (ETI) is a plant defense mechanism in which plant resistance proteins recognize the presence of effectors and initiate immune responses. Enhanced disease susceptibility 1 (EDS1) in Arabidopsis thaliana serves as a central node protein for basal immune resistance and ETI by interacting dynamically with other immune regulatory or resistance proteins. Recently, the effector HopA1 from Pseudomonas syringae was shown to affect these EDS1 complexes by binding EDS1 directly and activating the immune response signaling pathway. Here, we report the crystal structure of the effector HopA1 from P. syringae pv. syringae strain 61 and tomato strain DC3000. HopA1, a sequence-unrelated protein to EDS1, has an α+β fold in which the central antiparallel β-sheet is flanked by helices. A similar structural domain, an α/β fold, is one of the two domains in both EDS1 and the EDS1-interacting protein SAG101, and plays a crucial role in forming the EDS1 complex. Further analyses suggest structural similarity and differences between HopA1 and the α/β fold of SAG101, as well as between two HopA1s from different pathovars. Our structural analysis provides a foundation for understanding the molecular basis of the effect of HopA1 on plant immunity.





    Categories: Journal Articles
  • Corrigendum to “A clarification of the terms used in comparing semi-automated particle selection algorithms in cryo-EM” [J. Struct. Biol. 175 (2011) 348–352]
    [Apr 2015]

    Publication date: March 2015
    Source:Journal of Structural Biology, Volume 189, Issue 3

    Author(s): Robert Langlois , Joachim Frank







    Categories: Journal Articles
  • Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery
    [Apr 2015]

    Publication date: Available online 26 February 2015
    Source:Journal of Structural Biology

    Author(s): Gerlind Sulzenbacher , Véronique Roig-Zamboni , Willy J. Peumans , Bernard Henrissat , Els J.M. van Damme , Yves Bourne

    A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.





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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2









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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2









    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
    [Apr 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site
    [Apr 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    Author(s): Chathurada S. Gajadeera , Xinyi Zhang , Yinan Wei , Oleg V. Tsodikov

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg2+-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn2+-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn2+ at 2.1Å resolution. The active site contains two catalytic Mn2+ binding sites, each half-occupied, reconciling the previously observed 1:1 Mn2+:enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295–298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.





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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF
    [Apr 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • Progressive Stochastic Reconstruction Technique (PSRT) for cryo electron tomography
    [Feb 2015]

    Publication date: Available online 4 February 2015
    Source:Journal of Structural Biology

    Author(s): Beata Turoňová , Lukas Marsalek , Tomáš Davidovič , Philipp Slusallek

    Cryo Electron Tomography (cryoET) plays an essential role in Structural Biology, as it is the only technique that allows to study the structure of large macromolecular complexes in their close to native environment in situ. The reconstruction methods currently in use, such as Weighted Back Projection (WBP) or Simultaneous Iterative Reconstruction Technique (SIRT), deliver noisy and low-contrast reconstructions, which complicates the application of high-resolution protocols, such as Subtomogram Averaging (SA). We propose a Progressive Stochastic Reconstruction Technique (PSRT) – a novel iterative approach to tomographic reconstruction in cryoET based on Monte Carlo random walks guided by Metropolis–Hastings sampling strategy. We design a progressive reconstruction scheme to suit the conditions present in cryoET and apply it successfully to reconstructions of macromolecular complexes from both synthetic and experimental datasets. We show how to integrate PSRT into SA, where it provides an elegant solution to the region-of-interest problem and delivers high-contrast reconstructions that significantly improve template-based localization without any loss of high-resolution structural information. Furthermore, the locality of SA is exploited to design an importance sampling scheme which significantly speeds up the otherwise slow Monte Carlo approach. Finally, we design a new memory efficient solution for the specimen-level interior problem of cryoET, removing all associated artifacts.





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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2









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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2









    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
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    Author(s): Chathurada S. Gajadeera , Xinyi Zhang , Yinan Wei , Oleg V. Tsodikov

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg2+-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn2+-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn2+ at 2.1Å resolution. The active site contains two catalytic Mn2+ binding sites, each half-occupied, reconciling the previously observed 1:1 Mn2+:enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295–298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.





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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

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

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • Semi-automated selection of cryo-EM particles in RELION-1.3
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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
  • Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization
    [Feb 2015]

    Publication date: February 2015
    Source:Journal of Structural Biology, Volume 189, Issue 2

    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