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.





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  • 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
  • Characterization of the termini of the West Nile virus genome and their interactions with the small isoform of the 2′ 5′-oligoadenylate synthetase family
    [Apr 2015]

    Publication date: Available online 11 April 2015
    Source:Journal of Structural Biology

    Author(s): Soumya Deo , Trushar R. Patel , Grzegorz Chojnowski , Amit Koul , Edis Dzananovic , Kevin McEleney , Janusz M. Bujnicki , Sean A. McKenna

    2′ 5′-Oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding viral double-stranded RNA, OAS enzymes produce 2′-5′-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Truncations/mutations in the smallest human OAS isoform, OAS1, results in susceptibility to West Nile virus (WNV). We have previously demonstrated in vitro the interaction between OAS1 and the 5′-terminal region of the WNV RNA genome. Here we report that the 3′-terminal region is also able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3′-terminal region that is sufficient for activation of the enzyme. The solution conformation of the 3′-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3′-terminal region in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5′- and 3′-terminal regions, a required step for replication, is not sufficient to protect WNV from OAS1 recognition in vitro. These data provide a physical framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme.





    Categories: Journal Articles
  • Different Conformational Dynamics of Various Active States of β-Arrestin1 Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry
    [Apr 2015]

    Publication date: Available online 11 April 2015
    Source:Journal of Structural Biology

    Author(s): Dong Kyun Kim , Youngjoo Yun , Hee Ryung Kim , Min-Duk Seo , Ka Young Chung

    Arrestins have important roles in G protein-coupled receptor (GPCR) signaling including desensitization of GPCRs and G protein-independent signaling. Two major intra-molecular interactions, the polar core and the three-element region, maintain arrestins in the basal conformation by connecting the N- and C-domains. Mutations in these regions that disrupt the polar core (R169E or p44) or the three-element (3A) have been reported to interact with GPCRs in a phosphorylation-independent manner, and thus these mutants are referred to as pre-activated arrestins. On the other hand, deletion of 7 residues in the linker region between N- and C-domains (Δ7) freezes arrestins in the inactive state, which has a much lower binding affinity to GPCRs compared to the wild type form. Although these mutants are widely used for functional studies of arrestins, the conformations of these mutants have not yet been fully elucidated. Here, we analyzed the conformational dynamics of β-arrestin1 with various mutants (R169E, p44, 3A, and Δ7) by hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS data revealed that pre-activated mutants have more deuterium uptake than the basal state, and also that the regions and degree of increased deuterium uptake differ between pre-activated mutants. Unexpectedly, the inactive mutant also showed increased deuterium uptake in a few regions.





    Categories: Journal Articles
  • Structural basis for regulation of stability and activity in glyceraldehyde-3-phosphate dehydrogenases. Differential scanning calorimetry and molecular dynamics
    [Apr 2015]

    Publication date: Available online 11 April 2015
    Source:Journal of Structural Biology

    Author(s): Olga N. Makshakova , Pavel I. Semenyuk , Mikhail L. Kuravsky , Elena A. Ermakova , Yuriy F. Zuev , Vladimir I. Muronetz

    Tissue specific isoforms of human glyceraldehyde-3-phosphate dehydrogenase, somatic (GAPD) and sperm-specific (GAPDS), have been reported to display different levels of both stability and catalytic activity. Here we apply MD simulations to investigate molecular basis of this phenomenon. The protein is a tetramer where each subunit consists of two domains – catalytic and NAD-binding one. We demonstrated key residues responsible for intersubunit and interdomain interactions. Effect of several residues was studied by point mutations. Overall we considered three mutations (Glu96Gln, Glu244Gln and Asp311Asn) disrupting GAPDS-specific salt bridges. Comparison of calculated interaction energies with calorimetric enthalpies confirmed that intersubunit interactions were responsible for enhanced thermostability of GAPDS whereas interdomain interactions had indirect influence on intersubunit contacts. Mutation Asp311Asn was around 10Å far from the active center and corresponded to the closest natural substitution in the isoenzymes. MD simulations revealed that this residue had slight interaction with catalytic residues but influenced the hydrogen bond net and dynamics in active site. These effects can be responsible for a strong influence of this residue on catalytic activity. Overall, our results provide new insight into glyceraldehyde-3-phosphate dehydrogenase structure–function relationships and can be used for the engineering of mutant proteins with modified properties and for development of new inhibitors with indirect influence on the catalytic site.





    Categories: Journal Articles
  • Conical Fourier shell correlation applied to electron tomograms
    [Apr 2015]

    Publication date: Available online 3 April 2015
    Source:Journal of Structural Biology

    Author(s): C.A. Diebolder , F.G.A. Faas , A.J. Koster , R.I. Koning

    The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single axis tilt series acquisition. Acquisition of dual axis tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here we set out to determine whether it is beneficial fractionate electron dose for recording dual axis cryo electron tilt series or whether it is better to perform single axis acquisition. To assess the quality of tomographic reconstructions in different directions here we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce different anisotropic resolution in dual axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations.





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

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1









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

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1









    Categories: Journal Articles
  • Structural origin of the drastic modification of second harmonic generation intensity pattern occurring in tail muscles of climax stages xenopus tadpoles
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Gaëlle Recher , Pascal Coumailleau , Denis Rouède , François Tiaho

    Second harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation–contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy.





    Categories: Journal Articles
  • NMR structure and dynamics of Q4D059, a kinetoplastid-specific and conserved protein from Trypanosoma cruzi
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Aracelys López-Castilla , Tirso Pons , José R. Pires

    Q4D059 (UniProt accession number), is an 86-residue protein from Trypanosoma cruzi, conserved in the related kinetoplastid parasites Trypanosoma brucei and Leishmania major. These pathogens are the causal agents of the neglected diseases: Chagas, sleeping sickness and leishmaniases respectively and had recently their genomes sequenced. Q4D059 shows low sequence similarity with mammal proteins and because of its essentiality demonstrated in T. brucei, it is a potential target for anti-parasitic drugs. The 11 hypothetical proteins homologous to Q4D059 are all uncharacterized proteins of unknown function. Here, the solution structure of Q4D059 was solved by NMR and its backbone dynamics was characterized by 15N relaxation parameters. The structure is composed by a parallel/anti-parallel three-stranded β-sheet packed against four helical regions. The structure is well defined by ca. 9 NOEs per residue and a backbone rmsd of 0.50±0.05Å for the representative ensemble of 20 lowest-energy structures. The structure is overall rigid except for N-terminal residues A9 to D11 at the beginning of β1, K38, V39 at the end of helix H3 with rapid motion in the ps–ns timescale and G25 (helix H2), I68 (β2) and V78 (loop 3) undergoing internal motion in the μs–ms timescale. Limited structural similarities were found in protein structures deposited in the PDB, therefore functional inferences based on protein structure information are not clear. Q4D059 adopts a α/β fold that is slightly similar to the ATPase sub-domain IIB of the heat-shock protein 70 (HSP70) and to the N-terminal domain of the ribosomal protein L11.





    Categories: Journal Articles
  • Crystal structure and substrate-binding mode of GH63 mannosylglycerate hydrolase from Thermus thermophilus HB8
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): Takatsugu Miyazaki , Megumi Ichikawa , Hitoshi Iino , Atsushi Nishikawa , Takashi Tonozuka

    Glycoside hydrolase family 63 (GH63) proteins are found in eukaryotes such as processing α-glucosidase I and also many bacteria and archaea. Recent studies have identified two bacterial and one plant GH63 mannosylglycerate hydrolases that act on both glucosylglycerate and mannosylglycerate, which are compatible solutes found in many thermophilic prokaryotes and some plants. Here we report the 1.67-Å crystal structure of one of these GH63 mannosylglycerate hydrolases, Tt8MGH from Thermus thermophilus HB8, which is 99% homologous to mannosylglycerate hydrolase from T. thermophilus HB27. Tt8MGH consists of a single (α/α)6-barrel catalytic domain with two additional helices and two long loops which form a homotrimer. The structures of this protein in complexes with glucose or glycerate were also determined at 1.77- or 2.10-Å resolution, respectively. A comparison of these structures revealed that the conformations of three flexible loops were largely different from each other. The conformational changes may be induced by ligand binding and serve to form finger-like structures for holding substrates. These findings represent the first-ever proposed substrate recognition mechanism for GH63 mannosylglycerate hydrolase.





    Categories: Journal Articles
  • Presence of plicidentine in the oral teeth of the coelacanth Latimeria chalumnae Smith 1939 (Sarcopterygii; Actinistia)
    [Apr 2015]

    Publication date: April 2015
    Source:Journal of Structural Biology, Volume 190, Issue 1

    Author(s): F.J. Meunier , J. Mondéjar-Fernández , F. Goussard , G. Clément , M. Herbin

    The extant coelacanth Latimeria is a sarcopterygian predatory fish with caniniform teeth on its upper and lower jaws. The teeth are constituted of a cone of dentine with an apical cap of enamel, and they are fixed to the osseous component of the jaws by an attachment bone. Internal walls of the tooth base show folds that have been firstly interpreted in the past as radial vascular canals. Three-dimensional visualisation of these foldings using X-ray tomographic techniques and new histological interpretation lead to reconsider these structures as true plicidentine. The folds of the dentine do not invade the whole pulp cavity of the tooth contrary to the plicated condition of most fossil sarcopterygian fishes (e.g., Eusthenopteron, Porolepis, Megalichthys) certain fossil marine reptiles (ichthyosaurs) and extant varanids; in Latimeria they are limited to the lower third to the half of the pulp cavity. The presence of plicidentine in Latimeria’s teeth is proposed to be a plesiomorphic character for sarcopterygians.





    Categories: Journal Articles