A key challenge for volcanological science and hazard management is that few of the world’s volcanoes are effectively monitored. Satellite imagery covers volcanoes globally throughout their eruptive cycles, independent of ground-based monitoring, providing a multidecadal archive suitable for probabilistic analysis linking deformation with eruption. Here we show that, of the 198 volcanoes systematically observed for the past 18 years, 54 deformed, of which 25 also erupted. For assessing eruption potential, this high proportion of deforming volcanoes that also erupted (46%), together with the proportion of non-deforming volcanoes that did not erupt (94%), jointly represent indicators with ‘strong’ evidential worth. Using a larger catalogue of 540 volcanoes observed for 3 years, we demonstrate how this eruption–deformation relationship is influenced by tectonic, petrological and volcanic factors. Satellite technology is rapidly evolving and routine monitoring of the deformation status of all volcanoes from space is anticipated, meaning probabilistic approaches will increasingly inform hazard decisions and strategic development. Deformation is often used in volcano monitoring, but the associated hazard is hard to interpret. Biggs et al. analyse 20 years of global satellite data and show a strong statistical link between deformation and eruption, although deformation alone is not necessarily a precursor of imminent eruption. Volcanic unrest is characterized by changes in seismicity, deformation, gas emissions or fumarolic activityAlthough InSAR is burgeoning as a retrospective scientific tool for understanding magmatic plumbing systemsRather than relying on this small number of systems, we assess the global statistical relationships using remote observations. Although the multidecadal or shorter timescale of InSAR observations is short compared with many volcano repose periods, by observing a large number of volcanoes at different stages of their eruptive cycle, we adopt an ergodic assumption and substitute geographic coverage for catalogue time length. We then show how tectonic, petrological and volcanic factors influence this relationship.DE , but note that ‘predictive’ in this context refers to trait associations and does not imply deformation precedes eruption. The PPV of the contingency table  is 0.46. The NPV, that is, the proportion of ‘non-deformed’ volcanoes that did not erupt in the same period  and NPV (0.94–0.97) values.In order to obtain sufficient sampling across all categories, we use an additional 306 tropical volcanoes that were observed for the 3-year period 2007–2010 and select the corresponding 3-year period from the 198 volcanoes that were used for the global compilation. The PPV) for hotspot regions (0.33), basaltic (0.29) and andesitic compositions (0.34). Low volatile content and high magma supply rates contribute to the elastic storage of low compressibility magma in a shallow chamber for a short period before eruption.The proportion of eruptions is higher when there has been a historically recorded eruption (0.45), regardless of whether deformation was observed . DeformaPPV is lowest for rhyolite/dacite compositions (0.17), calderas (0.25) and rift settings (0.13). No phonolite or trachyte composition volcanoes deformed during 2007–2010, but the 18-year record shows a low PPV for this category too. Magmatic processes associated with the long-term storage of large magma volumes in the shallow crust may produce surface deformation without ensuing eruption 915The We find low NPVs for stratovolcanoes (0.94) and those with basaltic (0.93) and andesitic compositions (0.94) due to a high proportion of DE, and 12 as PPV value of 0.31 and a NPV of 0.92. Both these values are within the 95% confidence intervals of the PPV and NPV values for the unfiltered data set. Detailed investigation of the timescale of pre-eruptive deformation based on InSAR observations reveals the strong publication bias towards reporting periods of deformation. DE volcanoes and includes all eruptions for which more than 3 years of InSAR data are reported, excluding lava lakes, frequent eruptions (more than one every 3 years) and continuous effusion , meaning this information provides ‘strong’ evidential worthStatistical tests on ground-based data (primarily seismicity) indicate that pre-eruptive unrest duration varies between volcano types: from 2 days at complexPPV. For volcanoes with long eruption cycles, the satellite record tends to capture either deformation or eruption but rarely both. In the case of rhyolitic, dacitic, phonolitic and trachytic volcanoes and calderas, the inter-eruption period is characterized by shallow magmatic storage producing regular deformation episodes with infrequent eruptions, resulting in low PPVs. In the case of stratovolcanoes, the inter-eruption period is characterized by storage at a range of levels within a thick crust and compressible magmas, producing a high proportion of eruptions without deformation and a relatively low NPV. For volcanoes monitored using satellites alone and lacking field measurements, the variation in such diagnostic test statistics associated with globally mapped volcanological and tectonic factors could provide a solid footing for hazard analysis.Nonetheless, even with the current data set it is clear that, although the behaviour of individual plumbing systems is often poorly characterized, the statistics for some subsets of global volcanoes  phase delays caused by atmospheric water vapour, particularly the topographically correlated component, and (ii) the loss of signal coherence around snow-capped summits, dense vegetation or steep-sided edificesUnfortunately, the systematic studies are not uniform in either methodology or error reporting making it difficult to assess how many ‘hidden’ deformation signals exist. Some studies use individual interferograms31113331Volcanoes show episodic behaviour on timescales ranging between seconds to hundreds of thousands of yearsAlthough using the satellite archive allows us to consider a large set of volcanoes, the newness of the technology restricts the timescales observed. The current satellite-based deformation record spans 20 years in some temperate regions but only 3 years in the tropics  providin1937, D, is one where at least one period of deformation has been observed with InSAR, while ‘not deformed’, E, or ‘not erupted’, A volcano that ‘deformed’ormation . The subPPV to tend to 1; on shorter timescales, over which hazard decisions are made, the PPV is slightly greater than 0.4 for observation windows >9 years . The number of volcanoes that deformed and erupted rise as observation window length increases, with a particularly rapid increase over the first 3–6 years . Over ge>9 years .J.B. and S.K.E. carried out the analysis and J.B. wrote the paper with input from all authors. S.K.E., J.B. and T.A.M. conceived the systematic global approach. W.P.A. contributed to the statistical analysis; J.B., S.K.E., Z.L., M.E.P. contributed to the InSAR data set; J.B., R.S.J.S., S.K.E. and T.A.M. contributed to the interpretation.How to cite this article: Biggs, J. et al. Global link between deformation and volcanic eruption quantified by satellite imagery. Nat. Commun. 5:3471 doi: 10.1038/ncomms4471 (2014).Supplementary Tables 1-7 and Supplementary References


Glycoside hydrolases (GH) targeting cellulose, xylan, and chitin are common in the bacterial genomes that have been sequenced. Little is known, however, about the architecture of multi-domain and multi-activity glycoside hydrolases. In these enzymes, combined catalytic domains act synergistically and thus display overall improved catalytic efficiency, making these proteins of high interest for the biofuel technology industry.Actinospica, Hamadaea, Cystobacter, and Microbispora). Most of the proteins we identified contain a single catalytic domain that is frequently associated with an accessory non-catalytic domain. Regarding multi-domain proteins, we found that many bacterial strains have unique GH protein architectures and that the overall protein organization is not conserved across most genera. We identified 217 multi-activity proteins with at least two GH domains for cellulose, xylan, and chitin. Of these proteins, 211 have GH domains targeting similar or associated substrates , whereas only six proteins target both cellulose and chitin. Fifty-two percent of multi-activity GHs are hetero-GHs. Finally, GH6, −10, −44 and −48 domains were mostly C-terminal; GH9, −11, −12, and −18 were mostly N-terminal; and GH5 domains were either N- or C-terminal.Here, we identify the domain organization in 40,946 proteins targeting cellulose, xylan, and chitin derived from 11,953 sequenced bacterial genomes. These bacteria are known to be capable, or to have the potential, to degrade polysaccharides, or are newly identified potential degraders  contains supplementary material, which is available to authorized users. For example, most enzymes from GH families 5, 6, 7, 8, 12, 44, 45, and 48 act on cellulose, while GH families 10, 11, and 30 are mostly xylanases, and GH family 18, 19, and 85 are chitinases [Glycoside hydrolases (GH) are key enzymes for the processing of complex carbohydrates . Plant-dThe complete breakdown of polysaccharide requires the synergistic action of multiple enzymes acting on internal bonds , extremities , and intermediate degradation products . Thus, most identified degrader lineages have several genes coding for GH and many seemingly redundant enzymes targeting similar substrates , 7, 8. AA particular bacteria’s potential ability to deconstruct polysaccharides can be predicted by the number and diversity of GH domains in its genome , 13. In Our systematic investigation of the association and organization of catalytic and accessory domains involved in carbohydrate processing across sequenced bacterial genomes highlights new proteins, new domain architectures, and provide new insights about how bacteria are able to process complex carbohydrates with implications for biofuel research.We searched 11,953 sequenced genomes and identified 40,946 proteins containing 41,196 domains that target cellulose, xylan, or chitin . In contrast, some genera, although associated with known degraders, contained few GHs per genomes on average   contained 19 or 18 proteins with GH targeting cellulose, xylan and chitin. In these 2 genomes, most proteins were single-domain proteins targeting cellulose , xylan , and, in O. bacterium TAV5, two chitinases from GH18  than previously thought, and that many poorly characterized genera deserve further attention. However, several species from various genera were associated with >8 GHs of interest per genome   Fig. a. We alsExcluding unique domains, observed once, we identified 210 types of protein domains associated with the GH domains of interest  and Cytophaga (n = 4 genomes), the clustering based on protein architecture did not correlate with the distribution of GH domains. Next, in some genera, including Caldicellulosiruptor (n = 10 genomes), the clustering based on protein architecture correlated partially with the distribution of GH domains , the clustering of strains based on proteins targeting cellulose, xylan, chitin, and their accessory domains correlated with the clustering based on the distribution of GH domains  and, in a separate analysis, the distribution of GH domains only . However, we identified 217 proteins with multiple GH domains that targeted some combination of cellulose, xylan, or chitin . One hundred and five MA-GHs had 2 GH domains from the same family , whereas 112 MA-GHs had different GH domains  and avoids biased interpretation of bacterial carbohydrate processing based on known, or predicted, hydrolytic capabilities. To the best of our knowledge, the identified cellulases, xylanases, and chitinases described here outnumbered currently available databases (Table Actinospica) [Our HMM-based survey of bacterial genomes reveals the variability and the distribution of GH architecture in well-known degrader genera  , 13. Hownospica) ). In addnospica) , 13, 21.A. robineae DSM 44927 , increase the local concentration of catalytic domains  , and somStenotrophomas, Rhizobium, Gluconoacetobacter) displayed conserved protein architectures. In these genomes, knowing the exact protein architectures provides a way to estimate the GH content and the protein architecture in newly identified strains. However, beyond conserved sets of single-domain proteins, many bacteria display species-specific protein architectures. These unique protein architectures have no effect on the clustering of genomes and their domain organization cannot be predicted. This highlights the multimodularity of GH and suggests the rapid evolution of closely related organisms regarding their potential to target substrates in the environment. Thus, using our data, it is possible to infer the GH content of taxonomically identified bacteria and complex microbial communities . Because of the extensive variation even between closely related strains, however, inferring the exact protein architecture will remain a major challenge.Our analysis of the distribution of GH architectures across sequenced genomes supports the hypothesis that the distribution of GH domains and the potential for carbohydrate processing are taxonomically conserved , 9. ManyStreptomyces [Multi-activity proteins  mainly correspond to associations of GH domains targeting similar substrates . In addition, most MAGHs are homo-GHs . The association of two identical GH domains into MAGHs suggests a duplication-fusion of the catalytic domain, whereas the rare hetero-GHs  result from more complex recombination , 33. Thuptomyces  and filaptomyces ).C. bescii) produced unexpected enzymatic activity [Caldicellulosiruptor,  are essential for the deconstruction of complex carbohydrates  . The inc−5 and alignment coverage >60 % of PFam length were used in subsequent analyses. Substrate specificity of identified GH and CBM domains was derived from biochemically characterized bacterial homologs found in the CAZy database [https://www.patricbrc.org/portal/portal/patric/Home) [http://www.ncbi.nlm.nih.gov/Taxonomy/).Protein sequences from sequenced bacterial genomes were retrieved from the PATRIC database  and analdatabase , 7: GH 5ic/Home) . FinallyGH distribution and domain organization in sequenced bacterial genomes were analyzed using Vegan, Stats, and APE packages in the R software environment , 43. Clu


Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the ‘LHn fold’ across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.The botulinum neurotoxin type D is one of seven highly potent toxins produced by  Clostridium botulinum species are the causative agent of the rare neuroparalytic illness botulism. Seven distinct serotypes (A–G) of botulinum neurotoxins (BoNTs) affect humans and other species to varying degrees. Once inside the neuronal cell, BoNTs block the release of neurotransmitters leading to paralysisToxins from BoNTs are synthesized as a single polypeptide chain 150 kDa), which is post-translationally cleaved into a di-chain molecule composed of light chain  and heavy chain . LC is the catalytic domain and a zinc-endopeptidase, while HC is further divided into two sub-domains of equal molecular mass called the translocation domain (Hn) and the membrane binding domain (Hc). On binding to the nerve terminals, BoNTs are endocytosed into a vesicle, where the acidic environment causes some conformational changes allowing LC to enter the cytosol50 kDa, wet al.in vivo by electrophysiological study on human muscles8Of the seven types of botulinum toxin, A, B, E and F are known to cause the disease in human while C and D have only been observed in animal cases. More particularly, D has been responsible for several recent outbreaks of botulism in cattleE. coli and with a protease cleavage site located between the GHRH ligand and the LC, such that following protease activation to generate the active di-chain TSI the GHRH ligand is at the amino-terminus of the HC domain. This arrangement, termed central presentation, was necessitated by the requirement of the GHRH ligand to have a free N-terminus to be able to bind and activate its receptor. It means, however, that the relative arrangement of the binding domain relative to the LC and Hn domains is different to that in BoNT and previously reported TSI proteins14BoNT/A and /B are currently the only serotypes approved for medical uses. With the emergence of immuno-reactivity and resistance among patientsStructural and biochemical characterisation of the LHn fragments from serotypes A and B17P6422 with cell dimensions a = b = 173, c = 222 Å; α = β = 90, γ = 120° . The crystal of LHn/D diffracted at 2.3 Å resolution in space group γ = 120° . Despiteγ = 120° .2 contact area. Additionally LC and Hn (excluding the belt) make a further 1392 Å2 buried interface area principally involving hydrophobic interactions.LHn/D share 34 to 35% identity with the corresponding fragment of BoNT/A, /B and, /E, for which the full length structures are available192017The structure of the catalytically inactive mutant light chain superposes well with the high-resolution structure of a recombinant form previously reported  on one side (S70) and by the end of the ‘belt’ (F539) on the other (R63) . AdditioThe interaction between the catalytic domain and its VAMP substrate involves several binding sites based on the information provided by the structure of LC/F with a VAMP-inhibitor complexThe long substrate requirement of all BoNT serotypes is atypical to other zinc proteases and involves several binding sites distal from the catalytic pocketThe catalytic domain is normally linked to the translocation domain through a single disulphide bridge after activation of BoNT into a di-chain molecule. Although the construct used for crystallisation experiment was not activated, residues 442–457, corresponding to the engineered cleavage site and flexible linker between the two domains, were not visible. The cysteine bridge is however stabilised by formation of a small β-sheet between the C-terminus of LC, the N-terminus of Hn and the end of the belt segment .The belt region in /D is overall similar to the corresponding segments in serotypes /A, /B and /E . It surr2127The main components of the translocation domain are the two coiled-coil helices of approximately 105 Å . While tSXN101959 differs from LHn/D, and from natural BoNT molecules at the linker region between the two domains. It includes a Factor Xa cleavage site, a GHRH ligand domain (qGHRH[1–40]) and a GGGGS repeat . The engineered protein was crystallised as an activated di-chain molecule and diffracted up to 3.1 Å resolution . The twoAlthough the targeting ligand could not be observed, the crystal structure still provides valuable information on LHn/D as a framework for protein engineering. Indeed addition of a large peptide segment at the LC-Hn interface, which is essential for BoNT activityThe linker region (residues 438–513 in SXN101959) was engineered to provide ease of access of the targeting GHRH ligand for its receptor. It is flanked with an N-terminal protease cleavage site that allows activation of the whole molecule into its di-chain form, and frees the amino end of the peptide chain. The biological activity of GHRH is retained by the 29 N-terminal amino acids for recognition by the GHRH receptorIntegrity of the sample used for structural study was tested by measuring the intracellular accumulation of the second messenger cAMP in human GHRH receptor-expressing cells. SXN101959 elicited a concentration-dependent increase in cAMP accumulation with a pEC50 (potency) of 8.73, compared with the pEC50 of hGHRH(1–44) of 9.54. The maximum response of SXN101959 was the same as hGHRH(1–44), indicating that the TSI is a full agonist of hGHRH receptor. Activation of the target receptor confirms the presence of the qGHRH(1–40) ligand within the structure of SXN101959 . Thus thThe botulinum neurotoxin has been extensively studied by X-ray crystallography but little information is available on the relevance of these structures in solution. Small-angle X-ray scattering (SAXS) experiments were carried out on LHn/A, /B and /D. All data were collected on station X33 at DESY (Germany) and the solution scattering curves are provided in P(r)) in determining the radius of gyration (Rg) for each LHn serotypes with values of approximately 32, 34 and 34 Å for A, B and D, respectively (Dmax) of 110 Å, slightly longer than LHn/A at 102 Å. The comparable features of the three samples correlate with the known crystal structures. The theoretical scattering curves extrapolated from the high-resolution structures were calculated with CRYSOLRg were somewhat lower than the experimental ones (between 31 and 32 Å). Ab initio models were generated from the scattering curves  are some of the most potent protein toxins and their modular architecture have made them molecules of choice for the design of a novel class of biopharmaceuticals labelled Targeted Secretion Inhibitors. The intracellular activity of BoNT towards members of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor family, a critical element of vesicular secretion, can be redirected towards specific cell types. This concept relies on the functionality of the LHn fragment which consists of the catalytic and translocation domains of BoNT. The distinct but variable properties of each serotype, such as substrate specificity and duration of action, have led to the development of tailor-made TSI with optimised applicability. Recent report of the biological activity of SXN101959 in inhibiting GH secretion has demonstrated the significance of using LHn/D as a framework for protein engineering. The crystal structures of the LHn fragment and SXN101959 present for the first time the properties of the combined translocation and catalytic domains of BoNT/D. The data provided have highlighted the plasticity of the light chain and its extensive interaction with the translocation domain. The translocation domain appears as a ‘solid scaffold’ on which to build on a retargeting partner. Indeed the structure of SXN101959 showed that addition of a long peptide ligand in a central position did not cause any significant structural conformational changes, thus demonstrating the robustness of LHn structural framework, and its ability to accommodate novel arrangements of the domain subunits, such as central presentation, within TSI proteins. Finally, the solution scattering analysis confirmed the structural integrity of the LHn fragment across serotypes. Altogether this study has provided useful information on the structure of BoNT/D, and the LHn fragment in general, that will be essential for the development of the next generation of protein therapeutics based on the TSI technology.E. coli BL21 (DE3) cells. The LHn genes were engineered to encode for an enterokinase or factor Xa cleavage site between the LC and Hn domains. Proteins were purified by affinity chromatography using Ni2+-charged chelating sepharose column  and hydrophobic interaction . Samples were kept in 0.05M HEPES pH 7.2 with 0.2 M NaCl.Cloning, expression and purification were carried out following methods described previously17The LHn/D construct used for X-ray crystallography correspond to the catalytically inactive LHn/D double-mutant E230Q and H233Y. Standard site directed mutagenesis techniques employing PCR amplification and subsequent sequencing were used to create the mutated DNA construct. In addition to the mutations required to eliminate catalytic activity, this construct contains an engineered enterokinase cleavage site between LC and Hn but was kept as a single chain sample for crystallisation. Expression and purification were performed as per the other LHn samples described above.This protein was prepared as previously reportedH is the Hill slope. EC50 value is the negative log of the mid-point location of the curve.Frozen CHO-K1 cells recombinantly expressing the human GHRH receptor were rapidly thawed, collected by centrifugation and re-suspended in assay buffer Hams F-12 (Life Technologies) , 5 mM HEPES (Life Technologies), 1 mM 3-isobutyl-1-methylxanthine , 0.05% BSA (Perkin Elmer), pH 7.4). Cell suspension  was incubated in white 384-well microplates in triplicate with increasing concentrations of human GHRH  or SXN101959 (10 pM—1 μM), which had been serially diluted in buffer. Plates were incubated at 21 ± 3 °C for 90 minutes. Following ligand incubation, detection mix  (kit from Perkin Elmer) was added to each well. Plates were incubated at room temperature for 24 hours, and the fluorescence emitted at 665 and 615 nm following excitation at 320 nm was measured using an EnVision microplate reader (PerkinElmer). Raw data was plotted as 615/665 nm emission ratio and converted to percentage of the maximum response (Emax) defined by hGHRH(1–44). Dose response curves were fitted using a variable slope model (using GraphPad Prism software) using Crystals of LHn/D were grown with 2 μl of protein at 56 mg/ml mixed with 1 μl of reservoir solution consisting of 30% v/v pentaerythritol ethoxylate (15/4 EO/OH), 0.1 M HEPES 7.5, 6% w/v polyvinylpyrrolidone  and suspended above the well as a hanging drop. Crystals grew within 7–10 days into pear-like shapes of 50 to 500 μm in length.SXN101959 crystals were grown from the sitting drop method against 0.1 M sodium actetate pH 5.5, 0.8 M sodium formate, 10% w/v polyethylene glycol 8000, 10% w/v polyethylene glycol 1000 . A drop of 200 nl of protein at 10 mg/ml was mixed with equal amount of reservoir and incubated at 16 °C.1/2Diffraction data were collected at station I03 of the Diamond Light Source , equipped with a PILATUS-6M detector . A complete dataset to 2.3 Å and 3.1 Å were collected from single crystals at 100 K for LHn/D and SXN101959, respectively. Raw data images were processed and scaled with DIALSProtein samples of LHn/A, LHn/B, and LHn/D were kept in 0.05 M HEPES 7.2, 0.2 M NaCl for SAXS analysis and checked for monodispersity by dynamic light scattering . Samples were diluted to give three final concentrations at 5, 2.5 and 1 mg/ml. These dilutions were checked by absorbance measurement at 280 nm using a NanoDrop ND-1000 (Thermo Scientific), and concentration discrepancies were corrected for data analysis.P(r) analysis using GNOMab initio models that were averaged with DAMAVERSolution scattering (SAXS) data for the three LHn molecules were collected at DESY, (Germany), EMBL Hamburg, beamline X33How to cite this article: Masuyer, G. et al. Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors. Sci. Rep. 5, 13397; doi: 10.1038/srep13397 (2015).


The dielectric and ferroelectric behaviors of a ferroelectric are substantially determined by its domain structure and domain wall dynamics at mesoscopic level. A relationship between the domain walls and high frequency mesoscopic dielectric response is highly appreciated for high frequency applications of ferroelectrics. In this work we investigate the low electric field driven motion of 90°-domain walls and the frequency-domain spectrum of dielectric permittivity in normally strained ferroelectric lattice using the phase-field simulations. It is revealed that, the high-frequency dielectric permittivity is spatially inhomogeneous and reaches the highest value on the 90°-domain walls. A tensile strain favors the parallel domains but suppresses the kinetics of the 90° domain wall motion driven by electric field, while the compressive strain results in the opposite behaviors. The physics underlying the wall motions and thus the dielectric response is associated with the long-range elastic energy. The major contribution to the dielectric response is from the polarization fluctuations on the 90°-domain walls, which are more mobile than those inside the domains. The relevance of the simulated results wth recent experiments is discussed. P and its static/dynamic responses to electric field extE or/and other stimuli. In the mesoscopic level, the dielectric and polarization responses of a ferroelectric are determined by the specific domain structure and realized by the domain wall motions. In particular, the high-frequency applications of ferroelectrics have received continuous attention. A number of FE materials in thin film form have been used for high-frequency devices in microwave and optical communications as well as computing applications because of their high dielectric permittivity3Substantial efforts have been devoted to ferroelectric (FE) materials and their applications in advanced electronic technologies1The FE domain wall motion is a complicated process and depends on a series of intrinsic particulars such as defects, domain structures, and strains569103 and PbTiO3. First, epitaxial BaTiO3 and PbTiO3 thin films deposited on substrates such as SrTiO3, LaAlO3, and MgO, are often explored not only for fundamental understanding but also for potential applications, while textured polycrystalline films are also investigated. Second, these thin films offer regular twin-like (stripe-like) 90°-domain structure in coexistence with 180°-domains due to the intrinsic ferroelastic effects. In most cases, the 90° ferroelastic domains are dominant, and the order parameter is strongly coupled to the strains. Furthermore, the substrate induced strain imposes a competitive or coherent coupling with the internal ferroelastic strain in the films, making the domain structure and domain wall motion complicated. These behaviors, particularly in the high frequency range, are much less studied. It is believed that externally imposed strains have strong impacts on the domain wall motion and thus the high frequency dielectric permittivity1213Along this line, the most interested issue is the strain effect in perovskite FE oxide thin films with tetragonal lattice symmetry like BaTiOac electric field of high frequency, while the static responses have been well addressed. The dynamics of the domain wall may be characterized by the variation in dielectric permittivity as a function of the ac electric field (frequency ω and amplitude 0E), as long as the domain structure is well defined. A recent experiment demonstrating this dielectric response was carried out on clamped Pb(Zrx1-Tix)O3 (PZT) thin films deposited on Si substratesA prominent feature of the strain effects is the 90° domain walls vibration in response to an 3 or PbTiO3) and reveal the dynamics of the 90°-domain structure which couples the internal ferroelastic strains with externally imposed normal strains, in response to electric field extE. Instead of addressing the dynamic motion equation17182017While the strain coupling in polycrystalline PZT thin films may be too complicated, our purpose is to focus on tetragonal FE lattice  lattice with periodic boundary conditionsP(r) =  normalized by a pre-factor aPu(r) =  are imposed. We clarify that our simulation is not necessarily associated with a thin film, although the electric dipoles in our simulation can only relax on the in-plane configuration. An extension of the results to other geometry is plausible. For simplification, our simulation will not deal with the finite boundary problem since it will introduce complicated corrections to the mechanical balanceWe consider a tetragonal FE system as approached by an extE =  is confined on the lattice plane. The total free energy for this FE lattice can be written asldF, gF, ddF, elF, esF, and seF are the Landau potential, gradient energy, dipole-dipole interaction, elastic energy, electrostrictive energy, and electrostatic energy, respectively. Term ldF extending to the sixth-order is written as: A1, A11, A12, A111 and A112 are the Landau expansion coefficients and A1 = A10(T-T0). The lowest-order expression of term gF is: i,j = ∂Pi/∂rjP and jr = , and G11, G12, G44 and G′44 are the gradient energy coefficientsddF and seF can be written respectively as: ddF includes two contributions. One is the anisotropic part depF(0P) and the other is the isotropic part ddF′(δP) if we treat P(r) = 0P+δP(r) where 0P is the spatially homogeneous average polarization and r is the spatial vector. It is noted that 0P can be defined by: V is the volume of lattice.The electric field depF(0P) describes the depolarization energy and shares the same form as seF by difference of a 1/2 factor. In real system, the depolarization field is compensated by free charges for the cases with open boundaries. For the present case, no free charge is included. The effective electric field is independent of the depolarization field and simply equivalent to extE. In experiments, the electric field is usually applied via the electrodes in close-circuit which introduces free charge onto the interface. This will cancel out the depolarization field. Term ddF counts an integration over the whole lattice, and a realistic calculation is done either by the Fourier transformation or by finite truncation treatment27R is big (R = 8 in our simulation)2429Term elF yields: ijC is the elastic stiffness tensor which has only three independent elastic constants for a square lattice.The elastic energy esF is: q11 = C11Q11+2C12Q12, q12 = C11Q12+2C12(Q11+Q12), and q44 = 2C44Q44 are the effective electrostrictive coefficients, ijQ are the electrostrictive coefficients in external stress free state. Mathematically, we can also separate the total strain ijη into a homogenous component 0η, and a heterogeneous one ijδη which denotes the microscopic strain distribution at site 220η is imposed either by a substrate or an external load. Similar to generally accepted treatment2022u(r) is reduced to a function of P(r)ε(ω) over a broad range of frequency. The algorithms are presented in the Method section. The parameters for the simulations are listed in 32The electrostrictive energy dc) extE. To characterize the wall motion, we define the domain width l deviating from the equilibrium width l0 under zero electric field. Thus, Δl = l-0l stands for the offset in response to extE. In order to minimize the effect of domain wall irregularity, parameters l and 0l are calculated indirectly. A set of rectangle-like regions, whose two sides are on the domain walls to enable their areas as big as possible, are taken. We sum the areas of these rectangles. The local domain width l is obtained by dividing the area by its length and then performing statistics over sufficient number of rectangles in the same region.We first investigate the 90°-domain wall motion driven by a static  data are plotted in l(t) shows similar behavior and increases rapidly in the early stage and tends to be saturated at Δmaxl in the late stage. Nevertheless, Δmaxl depends on 0η, and the bigger 0η the smaller Δmaxl. In 0l(0η) for the a2-domain and corresponding Δmaxl(0η), showing the monotonous increasing of 0l(0η) and decreasing of Δmaxl(0η). For definition of each symbol and motion pattern of domain wall, one can refer to the In l(t) = a(1-ebt−) where a = Δmaxl and b are the fitting parameters. As shown in a and b as a function of 0η respectively imply that the a2-domain extension becomes tougher upon the transition from compressive strain to tensile one. It is also possible to evaluate the initial motion speed of the a2-domain walls by taking 0v = dΔl/dt|t→0 = ab, as shown in 0η<0), the motion speed of domain walls is higher than that for tensile strain (0η>0).The kinetics of wall motion was investigated earlier170η not parallel to extE, the above analysis is qualitatively correct. The tensile strain makes the domains with P//0η wider and the compressive one makes it narrower. This behavior allows a competition between the strain effect and electric field effect, and they can be coherent or cancelled depending on their orientation relationship. However, when the strain contains shear components, the situation becomes more complicated and extensive calculation is not discussed here.For the cases with strain ac-electric field, characterized by variation of dielectric permittivity as a function of 0η. In particular, the dielectric response in the frequency domain is related to the wall motion.Based on the above result, one understands that the 90°-domain structure clamped by external stress has different stability characteristics from the stress-free state. This difference can be discussed in the clamped 90°-domain structure driven by the 0η = 0. rε(f) and imaginary part iε(f) at three θ angles. Here a small 0E = 0.6|A1|aP is chosen, with the field direction defined by angle θ between the x-axis and extE. In general, rε(f) decreases gradually with increasing f for all the three cases, while iε(f) shows two peaks at characteristic frequencies Lf ~ 0.1τ−1 and Hf ~ 7τ−1, which are respectively referred as the low-f and high-f dispersions. Here τ = 1/|A1|D is the characteristic time for electric dipole flip (see the Method section for details). The striking feature is the low-f dispersion anisotropy, i.e. the θ-dependence which is the most remarkable at θ = 90° . This θ-dependence is shown by rε(θ) at f = 0.01τ−1 as an example in We first address the dielectric response in extE from θ = 0 to θ = 180°, one checks the domain evolution and dielectric dispersion. The low-f dispersion is related to the domain wall vibrations, while the high-f dispersion is attributed to the flip of individual dipoles. In fact, the dispersion peak around Hf is also observed in the mono-domain lattice.The above behaviors can be understood by investigating the instant evolution of domain structure. By turning f dispersion anisotropy in the domain scale. In 0η = 0, the spatial distributions of rε at f = 0.05τ−1 in four θ angles are presented in rε = 0.0~1.0). It is immediately seen that the dielectric permittivity mainly comes from the contribution of wall vibrations, while those dipoles deeply inside the domains contribute little. This characteristic makes the dielectric response very specific.Now we investigate the origin for the low-θ = 45°, the domain structure and dielectric response are shown in P·extE. The walls have the highest local mobility. No matter extE is positive or negative, there is always one of the neighboring two domains, inside which all the dipoles take the 135° angle from extE. This domain will shrink while the other will expand, making the wall move easily. This is also the reason why rε is the highest at θ = 45°, as seen in At θ = 90° (or θ = 0), as shown in rε is lower too. In this case, extE is parallel to the dipoles in one domain and perpendicular to those in the other. At θ = 112° and θ = 135°, as shown in rε. The wall mobility becomes the lowest at θ = 135°. rε except the very thin and dim lines on the walls. The reason is that term -P·extE in two neighboring domains are almost equivalent and they compete with each other, hindering the wall motion. The dielectric response is weak with no frequency dispersion.At f range and one example is given in f = 6τ−1 for two specific θ angles: θ = 90° and θ = 135°. The dielectric distribution over the whole lattice is roughly homogeneous. For details, one looks at the case in a1 and a2. The domain a1 whose dipoles align along the x-axis but perpendicular to extE shows slightly higher rε than that of domain a2. The reason is obvious that the dipoles in domain a1 are more fluctuating than those in domain a2.We finally check the dielectric response in the high-0η≠0. We only present in details the results on normally strained lattice. When the lattice is normally strained, the domain structure is deformed. To clarify the similarity and difference between the strain-free and strained lattices, we consider the simplest situation: 0η = 0.7% under extE with f = 0.05τ−1 and 0E = 0.6|A1|aP, both aligned along the y-axis (θ = 90°). We present in rε. For the two cases, the distributions are similar in amplitude but the high-rε spatial profiles across the domain walls are much wider for the strain-free lattice than those for the strained lattice. In the qualitative sense, the dielectric permittivity averaged over the whole strained lattice is lower than that over the strain-free lattice, at least in the low-f range. This 0η-dependence can be further illustrated in rε(f) and iε(f) at three tensile strains  are plotted. Both the real and imaginary parts are remarkably suppressed by tensile strain, and the Lf has a slight shift towards the high-f side. We also calculate the instant responses of the Δl and polarization component yP against extE at the three tensile strains, shown in l, yP, and extE, respectively, is observed. The responses of Δl and yP are synchronous with extE in the low-f range, and delayed in the high-f range. Furthermore, the wall vibration amplitude is suppressed by the tensile strain.Now we discuss the cases with 0η and extE are aligned along arbitrary directions independently, more complexity is seen in terms of the domain structure evolution and dielectric response. For some specific geometry, the strain and electric field may compete with each other. Some more discussion will be given below. However, in general, the calculated results are qualitatively similar to those for the simplest situation: the tensile strain suppresses the domain wall vibration, thus reducing the dielectric permittivity.The above results refer to the simplest situation. When 0η<0. Referring to the results under static (dc) extE, as shown in 0η-dependences of rε(f) in three compressive strains  along the y-axis are plotted in x-axis.Now we check the cases with extE is not parallel to 0η. One example is shown in 0η>0 and 0η<0, where extE is along the y-axis and 0η is along the x-axis. Our extensive calculations establish the qualitatively similar behaviors: the tensile strain suppresses the 90° domain wall motion and thus the dielectric permittivity, while the compressive strain enhances the domain wall motion and the dielectric permittivity, no matter whether extE is not parallel to 0η or not. We summarize spectrum rε in f<Lf, such as f = 0.01τ−1 and 0.05τ−1, rε falls gradually with increasing 0η from 0η<0 to 0η>0, while this tendency becomes negligible as f>Lf such as f = 0.5τ−1 since the single dipole response becomes dominant at this frequency. The overall evolution of rε is plotted in We also check the situations where It should be mentioned that the periodic boundary conditions allows the total strains in all directions to be exactly balanced out, and the strain effect is of long-range and coupled with mechanical boundary conditions. However, if other mechanical boundary conditions are considered, such as free boundary, the total strains can be partially relaxed through the free boundaries. The 90°-domain walls can be more moveable and thus show more significant response to electric field, as partially discussed in Ref. To understand the simulated results, one may give additional discussion by looking at the energy landscape and comparing the simulated results and experiments, although relevant experimental data are really rare.x-axis) is always accompanied with the tensile strain along the other direction (the y-axis), and verse vice. One can refer to the We calculate the elastic energy distribution associated with the 90° domain structures. For a single domain lattice, the total strain field is homogeneous, thus accommodating extremely large elastic energy. The single domain is decomposed into the 90° domain structure so that the total elastic energy can be relaxed. Owing to the lattice volume conservation, for one domain, the compressive strain along one direction  inside the a1-domain. Since the total elastic energy is proportional to η2, the rapidly enhanced total elastic energy inside the shrunk a1-domain acts as the resistant force against the electric field, responsible for eventual termination of the wall motion when the a1-domain becomes sufficiently narrow, as shown in the left column of elF is plotted. This explains why Δl tends to be saturated at maxΔl as time goes infinitive.First, we consider the 0η = 0.4% along the y-axis as an example. At t = 0 with extE = 0, the whole elastic energy distribution on the right column of 0η = 0. However, one can observe a much higher energy distribution in the shrinking a1-domains. Therefore, the a1-domains are already under highly tensile state along the x-axis even at extE = 0, when an externally imposed strain along the y-axis is applied. In this case, a static electric field along the y-axis may further force the wall moving into the a1-domain but will be highly resisted by the high strain energy, as shown in the right column of a1-domain driven by the electric field would become even tougher, given that 0η = 0.4% already makes the a1-domain narrow.This scenario applies to the lattice with externally imposed strain. Take 0η is applied along other orientations, no matter whether it is parallel to extE or not, the wall motion behaviors remain qualitatively similar. One example is given by applying extE along the x-axis where extE and 0η are normal to each other. As shown in 0η along the y-axis enhances remarkably the total elastic energy in the a2-domain, making the further shrinking of the a2-domain more difficult than the case with 0η = 0. Therefore, the above-discussed results don't lose the generality.If strain 3 (BTO) and Pb(Zr1-xTix)O3 (PZT) ceramics353611 Hz the real part of dielectric constant begins to drop, due to the frozen dipole oscillators themselves. In our simulation, the corresponding frequency is assigned as Hf = 7τ−1 in τ−1 ~1010 s−1, and the calculated Lf ~109 Hz.The implication of the above simulation data can be checked by comparing the simulated results with experimental data on the contribution of domain wall vibrations to the dielectric response in BaTiOA1|ε*)−1~103, where A1 is 3.8(T-479) × 105C−2m2N taken from PZT and ε* is the vacuum permittivity. The calculated real part of the dielectric permittivity at θ = 45° is ~200, as shown in x = 0.48–0.52) gave the difference in the real part between the extremely high frequency and the very low frequency, which is 250–500. The peak of the imaginary part is 150–265. These values agree roughly with our simulation results here. The above comparisons allow us to claim that the present calculations are reliable even in quantitative sense.Regarding the PZT ceramics, experimental data0.6Sr0.4TiO3 thin films upon increasing tensile strainWe also find some experimental results about the effects of tensile and compressive strains on the overall dielectric constant. In microwave frequency range, it was shown that dielectric constant and tunability of BTO films grown on MgO gradually decrease as the in-plane strain goes from the compressive type to tensile typet is time scaled in unit τ with τ−1 = |A1|D, and D is the kinetic coefficient. We also calculate the dielectric permittivity as a function of extE. For a dielectric system that cannot polarize instantaneously in response to an electric field, the total electric polarization as a function of t can be described as: P(t) is a convolution of electric field E(t) at previous times with time-dependent permittivity ε(t) = rε(t)+iiε(t). Therefore, its Fourier transformation can be directly written as P(ω) and extE(ω) are the Fourier transformations of P(t) and extE(t) respectivelyac sine electric field such as extE(t)0 = E·sin(ω0t) with ω0 = 2π0f and ω = 2πf, extE(ω) can be directly written as: E′0 is the coefficient of Fourier transformation and ω0 is the frequency. Polarization P(ω) can be calculated by Fourier-transforming the temporal evolution spectrum P in Eq.(10). In details, the real-spatial spectrum of P at site r and its spatial average P(t) over sufficient number of time periods is calculated by solving Eq.(10). The Fourier-transformed frequency spectrum P can be expressed as: P and P(t) into frequency domain to obtain P and P(ω). Subsequently, one can compute the corresponding dielectric permittivity ε at site r and its spatial average ε(ω0) at frequency ω0 using Eq.(12).The temporal evolution of the dipole lattice is tracked by solving the Ginzburg-Landau (TDGL) equation which takes the following form: J.M.L. conceived the research project and P.C. performed the computations. D.P.C., Y.L.W., Y.L.X., Z.B.Y., J.G.W. and J.Y.L. commented the modeling and discussed the results. P.C. and J.M.L. wrote the paper.Supplementary Materials


Apoptosis repressor with caspase recruiting domain (ARC) is a multifunctional inhibitor of apoptosis that is unusually over-expressed or activated in various cancers and in the state of the pulmonary hypertension. Therefore, ARC might be an optimal target for therapeutic intervention. Human ARC is composed of two distinct domains, N-terminal caspase recruiting domain (CARD) and C-terminal P/E (proline and glutamic acid) rich domain. ARC inhibits the extrinsic apoptosis pathway by interfering with DISC formation. ARC CARD directly interacts with the death domains (DDs) of Fas and FADD, as well as with the death effector domains (DEDs) of procaspase-8. Here, we report the first crystal structure of the CARD domain of ARC at a resolution of 2.4 Å. Our structure was a dimer with novel homo-dimerization interfaces that might be critical to its inhibitory function. Interestingly, ARC did not exhibit a typical death domain fold. The sixth helix (H6), which was detected at the typical death domain fold, was not detected in the structure of ARC, indicating that H6 may be dispensable for the function of the death domain superfamily. Cell death and proliferation must be balanced for multi-cellular organisms during developmental stages, as well as for homeostasis, and cells that present a threat to the integrity of an organism have to be removed. Apoptosis is a well-known mechanism for elimination of cells that maintains tissue homeostasis and protects organisms from accumulation of dangerous cells135791410BCL-2 family proteins such as BAX, BAK, BCL-2, BCL-XL, BCL-w, and BCL-B are required for regulation of the intrinsic cell death pathwayApoptosis repressor with caspase recruiting domain (ARC) is a multifunctional modulator of apoptosis. ARC is predominantly expressed in post-mitotic cells such as cardiomyocytes, neurons, and muscle cells, and unusually over-expressed in various cancers192+ in cytosol2+ can bind to the P/E domain of ARC, and Ca2+ depletion by ARC can block the apoptosis process29Human ARC contains 208 amino acids and is composed of two distinct domains, N-terminal caspase recruiting domain (CARD) domain and C-terminal P/E (proline and glutamic acid) rich domain . The CAR24252628To better understand the CARD domain-mediated inhibition mechanism of ARC during apoptosis signaling, we report the first crystal structure of the CARD domain of ARC containing amino acids 1–95 at a resolution of 2.4 Å. Our structure was a dimer and revealed novel homo-dimerization interfaces, which might be critical for the inhibitory function. In addition, the high resolution structure revealed similarities and differences with those of other CARD domains that may be functionally relevant. Interestingly, ARC did not exhibit a typical death domain fold , and instead contained a five-helix bundle fold. H6 was not detected in the structure of ARC, indicating that it is dispensable for the function of the death domain superfamily.ARC inhibits both the intrinsic and extrinsic apoptosis pathways via interaction with various apoptosis related proteins, including Fas, FADD, and caspase-8 . The ARCwork of 17.0% and an Rfree of 20.0%. The high resolution structure of ARC CARD showed that it comprises five helices, H1 to H5, which is not the typical fold of the DD superfamily  was solved using a single-wavelength anomalous diffraction (SAD) method and refined to an Rerfamily . Intereserfamily . The moderfamily . Each moerfamily . Althougerfamily . The puterfamily . Residueerfamily . The higfamily , which represents 9% of the dimer surface area. The principal interaction forces are salt bridges and hydrogen bonds, which are made by D13 (on H1), R56 (on H4), R59 (on H4), and R60 (H4) from chain A and by D32 (on H2), R37 (on H2), E46 (on H3), and D49 (on H3) from chain B (1433Size-exclusion chromatography and multi-angle light scattering (MALS) indicated that the isolated ARC CARD behaves as a dimer in solution . The cal2433 chain B . At the To analyse the novel homo-dimeric structure of ARC CARD formed by large electrostatic interactions, we conducted size exclusion chromatography and MALS experiments in the presence of high salt (1.5 M NaCl) and low pH (pH 3), which resulted in the carboxylates being protonated and the charged interactions disrupted. As expected, ARC CARD became a monomer at high salt and low pH conditions  and 2B. Taken together, ARC CARD forms homo-dimer in solution, as expected, which was shown in a previous in vivo experimentA structural homology search with DALIPair-wise structural alignments between ARC CARD and these other CARDs showed clear structural differences and similarities . The CARARC CARD has similar gross features in its electrostatic surface as other CARDs. For example, similar to the charged nature of most other CARDs, the ARC CARD surface is also composed of a mixture of positively and negatively charged features. The charged clusters are located in the interface of the homo-dimeric complex. Because CARDs are protein interaction modules, their surface features dictate their mode of interactions with partners. Based on analysis of the electrostatic surface of ARC CARD, it might be possible to use this charged interaction for homo or hetero dimeric complex formation.A number of residues on the surface of the ARC CARD that have been identified as critical to formation of the interface of the homo-dimeric complex are conserved on Fas (DD), FADD (DED), and caspase-8 (the first DED), which are known binding partners of ARC . These iThree types of interactions  at six unique interfaces  have been identified in the interactions with the death domain superfamily 1438. The14Based on the sequence alignment, previously solved CARD complex structure, and location of the conserved residues critical to formation of the homo-dimeric complex of ARC CARD, we developed a tentative inhibitory model of ARC CARD by interacting with Fas DD or FADD DD. Because D13, R56, R59, and R60, which are critical residues for the formation of one side of the homo-dimeric interface of ARC CARD, are not relatively conserved at the Fas DD and FADD DD, this side of ARC CARD was used as a tentative interaction site. Fas DD or FADD DD was then superimposed on the ARC CARD located on the other side to make a Type I interaction. The ARC:Fas complex model showed that R56, R59, and R60 from ARC CARD form a massive charged interaction with D261 and E256 from Fas DD . D13 of The first homo-dimeric CARD structure of ARC and follow-up studies suggest an ARC-mediated molecular inhibitory mechanism of the DISC assembly, which is the critical signalling molecular complex in the extrinsic apoptosis signalling pathway. Since the multimeric CARD domain complex of RIG-1 and MAVS, which use all three types of interactions that have been detected for assembly of the death domain subfamily, were recently reported, the fact that ARC CARD forms a higher oligomeric complex with Fas DD or FADD DD during the inhibition process cannot be ignored. In this case, an extensive surface of ARC CARD with residues spreading through all five helices of its structure might have participated in assembly of the inhibitory complex. Further efforts to solve the complex structure are needed to obtain clearer insight into the molecular basis of inhibition by ARC. However, it has been reported that homo-dimerization of ARC CARD caused loss of inhibitory activity of ARC, indicating that our proposed model of the complexes with Fas DD and FADD is accurate. Revealing the protein interaction interface detected in the current study might be critical to the interaction with other binding partners for the proper inhibitory function of ARC during apoptosis and necroptosis.ARC is prominently expressed in cardiac and skeletal muscle cells and prevents apoptotic cell death of those post-mitotic muscle cells. Involvement of ARC in cardiomyopathy under stress conditions was observed in a mouse modelE. coli BL21 (DE 3) under overnight induction at 20°C. The protein contained a carboxyl terminal His-tag and was purified by nickel affinity and size exclusion chromatography using a size 200 exclusion column 10/30  that had been pre-equilibrated with a solution of 20 mM Tris-HCl at pH 8.0 and 150 mM NaCl. The protein was then concentrated to 8-9 mg/ml for the crystallization trial.The expression and purification methods used in this study have been described in detail elsewhere. Briefly, ARC CARD domain (amino acids 1–95) was expressed in -1 protein in 20 mM Tris-HCl at pH 8.0, 150 mM NaCl) and 1 μl of a reservoir solution containing 0.6 M ammonium phosphate and 0.1 M imidazole (pH 8.2) against 0.3 ml of reservoir solution. Selenomethionine-substituted ARC CARD was produced using a previously established methodThe crystallization conditions were initially screened at 20°C by the hanging drop vapour-diffusion method. The final diffractable crystals were grown on plates by equilibrating a mixture containing 1 μl of protein solution  method was conducted using Phaserhttp://www.ebi.ac.uk/Tools/clustalw2/index.html).Amino acid sequences were analysed using Clustal W  that had been equilibrated in appropriate buffer. The chromatography system was coupled to a three-angle light scattering detector (mini-DAWN EOS) and a refractive index detector (Optilab DSP) (Wyatt Technology). Data were collected every 0.5 s at a flow rate of 0.2 ml/min and then analyzed using the ASTRA program, which gave the molar mass and mass distribution (polydispersity) of the sample.Site-directed mutagenesis was conducted using a Quick-change kit (Stratagene) according to the manufacturer's protocols. Mutagenesis was then confirmed by sequencing. Mutant proteins were prepared using the same method as described above.Changes in oligomerization in response to mutations were monitored by native (non-denaturing) PAGE using a Phast System  with pre-made 8–25% acrylamide gradient gels . Separately purified proteins were pre-incubated at room temperature for 1 hour before loading the gel. Coomassie Brilliant Blue was used for staining and detection of shifted bands.The secondary structures were measured by circular dichroism (CD) spectroscopy using a J-715 spectropolarimeter at the Korea Basic Science Institute in South Korea. The spectra were obtained from 200 to 250 nm at 25°C in a 0.1 cm path length quartz cuvette using a bandwidth of 1.0 nm, a speed of 50 mm/min, and a 5s response time. Four scans were accumulated and averaged, after which the α-helical content was calculated from the molar ellipticity at 222 nmHow to cite this article: Jang, T.-h. et al. Crystal structure of caspase recruiting domain (CARD) of apoptosis repressor with CARD (ARC) and its implication in inhibition of apoptosis. Sci. Rep.5, 9847; DOI:10.1038/srep09847 (2015).Accession Codes: Coordinates and structural factors have been deposited in the RCSB Protein Data Bank. The PDB ID code is 4UZ0.


Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N6-methylated adenosine reader domain and report its solution structure in complex with a N6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m6A recognition. These findings establish a molecular function for YTH domains as m6A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m6A recognition.N Two studies used RNA immunoprecipitation and mass spectrometry to identify proteins binding selectively to the m6A-containing RNA sequences   Figure , which w) Figure ,15. The ) Figure . Subsequ) Figure . A more ein MMI1 ,19.6A recognition by the YTH domain. On the basis of the structure, we rationalize why more generally YTH domains act as reader domains for m6A.Here we show that the YTH domain of YT521-B binds sequence-specifically a GA-containing sequence with a 50-fold affinity increase when the adenine is N6 methylated. We determined the solution structure of the complex which provides the structural basis of the specific mR. Norvegicus YT521-B protein were cloned into the pTYB11 vector (New England Biolabs). The used cloning scheme results after intein cleavage in a protein without any vector-derived residues. Escherichia Coli BL21 (DE3) codon plus (RIL) cells were grown in M9 minimal medium containing 1 g/l 15NH4Cl and 4 g/l unlabeled glucose (15N-labeling) or 2 g/l 13C-labeled glucose (13C15N labeling). Protein expression was induced at a cell density of A600 ≈ 0.6 by addition of 1mM isopropyl-β-D-thiogalactopyranoside. After induction the temperature was reduced from 37°C to 18°C. After overnight expression cells were harvested, resuspended and lysed by cell cracking. Protein purification on chitin columns and intein-mediated cleavage was performed according to the manufacturer's instruction. For cell lysis and intein purification a buffer containing 20 mM Na2HPO4, 0.5 M NaCl at pH 8 was used. For intermediate washing steps, a buffer containing 1 M NaCl instead of 0.5 M NaCl was used. For cleavage the lysis and intein purification buffer contained in addition 50 mM DTT. After the elution of the YTH domain from the chitin column, the eluate was concentrated with 5 kDa cutoff centricons (Vivaspin) and purified with size exclusion chromatography (SEC) using a Superdex 75 column  in a buffer containing 25 mM NaH2PO4, 25 mM NaCl and 10 mM β-Mercaptoethanol at pH 7 (pH adjusted with HCl) with the addition of 200 units of SUPERase In RNase inhibitor (Ambion) prior to loading of the sample on the SEC column. Fractions containing the YTH domain were pooled and concentrated for further experiments. RNAs were deprotected according to the manufacturer's instructions (Thermo Fisher), lyophilized and resuspended in the size exclusion buffer.Residues 347–502 of the 6ACAC-3′ were performed at a concentration of 0.8 mM at 30°C. Nuclear magnetic resonance (NMR) measurements were carried out in size exclusion buffer either containing 90% H2O/10% D2O or 100% D2O. Spectra were acquired on Bruker AVIII 500 MHz, AVIII 600 MHz, AVIII 700 MHz and AVIII HD 900 MHz spectrometers equipped with cryoprobes. Data acquisition and processing was carried out with Topspin3 (Bruker). NMR data were analyzed with Sparky 3.114 .Protein RNA titrations were measured with a protein concentration of 0.2 mM and at a temperature of 20°C. Measurements for the structure calculation of the YTH in complex with 5′-UGm1H-15N HSQC, 2D 1H-13Caliphatic HSQC, 2D 1H-13Caromatic HSQC, 3D HNCO, 3D HNCA, 3D HNCACB, 3D CBCACONH, 3D 13C-15N-1H HCC(CO)NH-TOCSY, 3D 1H-15N-1H HCC(CO)NH-TOCSY, 3D-NOESY 1H-15N HSQC (τm = 150 ms), 3D NOESY 1H-13Caliphatic HSQC (τm = 150 ms), 3D NOESY 1H-13Caromatic HSQC (τm = 150 ms). All were collected in buffer containing 90% H2O/10% D2O. For observation of slowly exchanging amide protons a 2D 1H-15N HSQC in 100% D2O was recorded. For RNA resonance assignment the following spectra were used: 2D 1H-1H NOESY (τm = 150 ms), 2D 1H-1H TOCSY (τm = 60 ms), 2D F1-filtered F2-filtered 1H-1H NOESY (τm = 150 ms)  peak assignment a 3D 13C F1-edited F3-filtered NOESY 1H-13C HSQC (τm = 100 ms) collected in 100% D2O and a 2D F2 filtered 1H-1H NOESY (τm = 150 ms) recorded in 90% H2O/10% D2O were used (For protein resonance assignment the following spectra were used: 2D  150 ms) , naturalere used .6A. Restraints for backbone dihedral angles based on chemical shifts were obtained using the program Talos+ . The electrostatic surface potential was generated with the PyMOL APBS Tools plugin using PDB2PQR 2 + (δN/6.51)2]1/2. Peaks in intermediate exchange, which could not be followed over the course of the titration, were assigned a perturbation value of 0.3 ppm. Perturbations for each oligonucleotide assayed were summed up and for each position normalized to the one with the largest perturbation sum.In contrast to the original scaffold-independent approach  in the mK, ΔH, ΔS and N) with respective errors  were determined based on χ2 minimized fit of the experimental data to a single-site binding model as implemented in the Origin software version 7 (Origin Lab) provided with the VP-ITC instrument. For the binding of 5′-UGACAC-3′, 410 μM of RNA has been injected into 14 μM of YTH. For the interaction with 5′-UGm6ACAC-3′, we used 125 μM and 10 μM of RNA and protein concentrations, respectively. The isothermal titration calorimetry (ITC) experiments were set to deliver 45 (6 μl) injections at 300 s intervals. Stirring speed and reference power were 307 rpm and 10 μcal/s, respectively. For each control, heats of dilution were not significant .Affinity measurements by isothermal titration calorimetry (ITC) were performed at 20°C using a VP-ITC calorimeter  in identical buffer conditions as described previously for the SEC step. Thermodynamic parameters  for YT521-B allowed us to map the RNA-binding surface of the YTH domain, the fact that many protein resonances were absent in the spectra of the complex prevented us to determine its structure . We thereviously ,10–11. T6A3 showed that the complex formation shifted from a fast/intermediate to a slow exchange regime clearly indicating an increase in binding affinity mediated by the m6A modification  that interact with the phosphate oxygens are strictly conserved among YTH domains (Lys 364 and Arg 478) suggesting that most YTH domains could recognize specifically m6A-containing RNA and bind in a very similar manner as YT521-B. Based on our structure, we built homology models of the human YTHDF1 YTH domain and the Saccharomyces cerevisiae MRB1 YTH domain which were previously shown to bind m6A-containing RNA , which is conserved in YTHDF2 and 3  reveals that most of the residues interacting with the RNA are conserved  demonstH domain 6A are not exception to this theme despite the fact that the m6A modification does not create a positive charge unlike m7G for example , this finding could have a major biological implication. It was recently found that YTH domain-containing proteins are found in the multi-subunit protein complexes responsible for m6A RNA modification (6A sites contain a GA sequence.It was quite unexpected to find that the YTH domain of YT521-B has a sequence-specificity for GA-containing RNA (even without adenine methylation). Although this affinity is modest  with accession code 2MTV. Chemical shifts and restraints used for structure calculation were deposited in the Biological Magnetic Resonance Data Bank (www.bmrb.wisc.edu) with accession number 25188.Structural coordinates were deposited in the PDB database (Supplementary Data are available at NAR Online.


Coxiella burnetii or Rift Valley fever virus.Following the emergence of the Bluetongue virus serotype 8 (BTV-8) in France in 2006, a surveillance system (both passive and active) was implemented to detect and follow precociously the progression of the epizootic wave. This system did not allow a precise estimation of the extent of the epizootic. Infection by BTV-8 is associated with a decrease of fertility. The objective of this study was to evaluate whether a decrease in fertility can be used to evidence the under-reporting of cases during an epizootic and to quantify to what extent non-reported cases contribute to the total burden of the epizootic. The cow fertility in herds in the outbreak area (reported or not) was monitored around the date of clinical signs. A geostatistical interpolation method was used to estimate a date of clinical signs for non-reported herds. This interpolation was based on the spatiotemporal dynamic of confirmed case herds reported in 2007. Decreases in fertility were evidenced for both types of herds around the date of clinical signs. In non-reported herds, the decrease fertility was large (60% of the effect in reported herds), suggesting that some of these herds have been infected by the virus during 2007. Production losses in non-reported infected herds could thus contribute to an important part of the total burden of the epizootic. Overall, results indicate that performance data can be used to evidence the under-reporting during an epizootic. This approach could be generalized to pathogens that affect cattle’s performance, including zoonotic agents such as  Following the emergence of the Bluetongue virus serotype 8 (BTV-8) in France in 2006, a surveillance system was implemented to detect and follow precociously the progression of the epizootic wave. This system was composed of both passive  and active surveillance (blood sampling and diagnostic tests in targeted populations). This system did not allow a precise estimation of the extent of the epizootic. A cross-sectional serologic study conducted in 2007 showed that only a low proportion of seropositive herds reported clinical cases, indicating a high under-reporting rate of clinical cases Infection by BTV-8 is associated with a decrease of fertility In most dairy cattle, artificial inseminations (AI) are used for reproduction. This information on individual cow reproduction is centralized in a national data base. The monitoring of these events can be used to evaluate reproduction performance at the cow level. For example, the occurrence of repeated AI can be used as proxy of cow fertility disorders Decrease in fertility could be used as non-specific indicator to evaluate the existence of under-reporting of BTV-8 cases in exposed areas. Moreover, quantifying such losses in non-reported herds would enable to obtain a more comprehensive evaluation of the burden of an epizootic in a newly infected area than when accounting only for case herds. The objective of this study was to evaluate whether a decrease in fertility can be used to evidence the under-reporting of cases during an epizootic and to quantify to what extent non-reported cases contribute to the total burden of the epizootic.Decrease in fertility was quantified for cows in reported case herds and cows in non-reported herds located in the 2007 outbreak area (herds with uncertain infectious status). These quantifications were performed using cows in herds unexposed to BTV-8 in 2007 as reference population.Information about exposure of herds to BTV-8 during 2007 was obtained from the official veterinary surveillance system. Herds were reported in 2 distinct situations: (i) in the event of clinical signs detected by the farmer or the veterinary practitioner, subsequently confirmed by a diagnostic test and (ii) in the event of a positive serological test performed either before animal transfer or sale, or in sentinel herds. Among herds reported during 2007, only herds with a confirmed detection reported after clinical suspicion (situation (i)) were included as the date of a positive serological test did not necessarily identify the possible date of BTV-8 exposure of the herd. Information about BTV-8 exposure was available at the herd level only. Thus, a herd was considered exposed if at least 1 animal with clinical signs had tested positive for BTV-8. The proportion of infected animals in reported herds was unknown. Herds reported after clinical suspicion and subsequently selected for this study will be referred to as ‘case herds’. Case herds located in 23 departments were selected , corresponding to 96% of all cattle herds reported due to clinical suspicion during the 2007 epizootic. Cattle herds that were not reported during 2007 and located in these departments were considered to have an uncertain infectious status, and were thus referred as ‘herds with uncertain BTV-8 status’ .The performance data were obtained in dairy herds enrolled in the official Milk Recording Scheme and where artificial insemination was used. For each cow, obtained data were dates of AIs, rank of service (number of AI within lactation), date of culling (if it had occurred during the study period) and data used to adjust for factors known to influence fertility: cow and bull breeds, lactation number, calving date, milk production data at each milk record . After selection of dairy herds that use AI for reproduction, populations of case herds and herds with uncertain BTV-8 status were composed of 4,392 and 13,804 herds respectively.A date of exposure to BTV-8 was either estimated from recorded data for cows in case herds, or interpolated for cows in herds with uncertain BTV-8 status. This interpolation was based on the spatio-temporal dynamics of detection of confirmed case herds that reported clinical signs in 2007. Decrease in fertility in both case herds and herds with uncertain BTV-8 status were quantified around the date of exposure (observed or predicted). The geographical coordinates of herds were available at the municipality level.For each case herd, available data included the date at which clinical signs of disease were first suspected and the date at which disease was confirmed via diagnostic tests. The estimated date of exposure for reported herds was defined as the recorded date of suspicion which corresponded to the first detection of clinical signs in the herd. The same date of exposure was assigned to all cows in a herd. For 6.1% of the case herds, the date of clinical suspicion was missing but the date of confirmation by a diagnostic test was known. In order to assign a date of suspicion, an imputation method based on the distribution of the time interval between dates of suspicion and confirmation was applied . Moreover, 181 case herds that had a non-plausible time interval between dates of suspicion and confirmation  were excluded.A date of exposure to BTV-8 for each cattle herd with uncertain BTV-8 status during 2007 was interpolated.Culicoïdes. Densities of beef and dairy herds widely vary among French areas. This method is based on assumptions regarding the form of the trend of the sample data, its variance and spatial correlation. The first step consisted in modelling the spatial correlation of the data. Two models were compared. A cross-validation process with observed data was used to determine each model’s goodness of fit and to compare their predictions. Spatial variation in detection dates were modelled using a gaussian semivariogram – for smooth long-range propagation waves - and an exponential semivariogram - for short-range random propagation between neighbouring herds - models. To account for the non stationarity of the BTV-8 spreading process, the gradient of the viral diffusion was also included in the model by the use of Universal Kriging in place of Ordinary Kriging. For the final interpolation of detection dates, only the gaussian-model spatial component was kept for filtering the random local component.Kriging, a geostatistical interpolation method, was used to estimate a date of detection of clinical signs for herds with uncertain BTV-8 status. Dates were expressed as the number of days since the first case herd reported in 2007. Kriging uses data sample (cattle case herds) to predict values at unsampled locations (herds with uncertain BTV-8 status). All the cattle herds (dairy and beef) were included because they could have all played a role in the epizootic wave diffusion. Both beef and dairy herds are exposed to A reference population was used to quantify both decrease in fertility of cows in case herds and cows in herds with uncertain BTV-8 status. It was composed of cows located in 2 French regions unexposed to BTV-8 during 2007: Brittany and a south-western area. In France, herd management varies between areas. Therefore, unexposed cows from 2 different parts of France were selected in order to better represent the unexposed population. This comparison limited the impact of any possible confounding factors due to variations of herd management over time.The month of AI is known to influence fertility Fertility was assessed by the occurrence of a repeat AI after the first AI. In case of a return-to-oestrus after the first AI in a lactation, most farmers are likely to decide to reinseminate the cow. In such a context, the occurrence of a repeat AI (return-to-service) after a first AI can be used as a proxy for infertility. The criteria used to quantify effect of BTV-8 exposure on fertility was the 90-d-return-to-service, defined as a return-to-service occurring between 18 and 90 d after AI (binary variable). It was assumed that BTV-8 exposure could have 3 possible effects on fertility: conception failure, embryonic death and fetal death in early gestation, all resulting in the same outcome. Cows with non-plausible or extreme data  and herds with unusual management  were excluded. After these exclusions, the study population was composed of 122,079 cows with a first AI performed in 2007  in 7,883 herds located in the epizootic area . The reference population was composed of 211,578 cows in 9,485 herds located in regions unexposed to BTV-8.To evaluate the under-reporting of BTV-8 exposure, 90-d-return-to-service of cows in case herds and cows in herds with uncertain BTV-8 status were compared to those of cows in the reference population. All first AIs performed from 10 weeks before to 14 weeks after the date of BTV-8 exposure (observed or predicted) in herds were selected. The AIs were classified into categories according to the time interval between the date of the AI of cows and the date of exposure (observed or predicted). Intervals of 15 d were considered (see results for categories).t for the probability of 90-d-return-to-service following the first AI for the ith cow in the jth herd, X is the vector of the 6 following fixed effect variables: the exposure status (36 classes corresponding to 12 periods of 2 weeks for 3 populations); the lactation number (4 classes); the maximum milk production in kg at the 3 first milk records in the lactation used as a proxy for peak milk yield (5 classes); the minimum of Protein:Fat ratio out of the first 3 milk records (5 classes); the calving-to-AI interval (7 classes) and the month of AI (8 or 10 classes), The relationship between exposure and occurrence of a possible return-to-service was assessed with multivariate Cox models. To account for factors likely to influence the probability of return-to-service, the association between BTV-8 exposure and occurrence of return-to-service was adjusted for several independent variables already described as risk factors for fertility traits in the literature To check whether decreases in fertility in both case herds and herds with uncertain BTV-8 status can be attributable to BTV-8 exposure, possible decrease was also quantified for cows in herds that were not exposed to BTV-8. Indeed, decrease could be related to the climate or diet. This population was considered to demonstrate the specificity of the fertility decreases regarding the BTV-8 exposure, reported or not. This population was composed of cows inseminated in 2005 in herds that were located in the 2007 outbreak area (reported or not). The date around which fertility decreases were quantified corresponded to the same day within the year  than the date of exposure (observed or predicted). This population (after the same exclusions than the exposed and unexposed populations) was composed of 211,578 cows with a first AI in 9,485 herds.Cattle case herds reported after clinical signs in 2007 were located in the north-eastern part of France . The ove2 and a fitted exponential semivariogram model with a semi-variance of 127 day2 and a range of 9.9 km. The fitted gaussian semivariogram model had a semi-variance of 243 day2 for a scale parameter (sd) of 82 km that is equivalent to an effective range of about 160 km. The fitted nested semivariogram model, plotted in black in st July 2007 among cattle herds.As expected, exposure to BTV-8 at the time were clinical signs were observed was associated with an increase of 90-d-return-to-service rate for cows in case herds . The perThe following adjustment variables were significantly associated with a risk of 90-day-return-to-service: lactation number, milk yield, Protein:Fat ratio and calving-to-AI interval. The risk was higher for multiparous cows and for cows with a higher milk yield. In contrast, the risk was lower for cows with a higher Protein:Fat ratio and for cows with a higher calving-to-AI interval .The results indicate that fertility data can be used to evidence the under-reporting of cases during an epizootic. Indeed, an decrease in fertility was found for cows in non-reported herds located in the 2007 outbreak area in France, corresponding to 60% of the effect for cows in case herds. Only a very slight decrease in fertility was found when comparing two populations of cows in unexposed herds, indicating that the episodes of fertility decreases quantified in this study were likely attributable to BTV-8 exposure. Moreover, the fertility decreases were precocious relative to the date of clinical suspicion.The magnitude and duration of the episode of fertility decrease for cows in non-reported herds suggested that some of these herds have been infected by the virus during 2007. If assuming a similar average effect of infection between case herds and non-reported herds, around 60% of non-reported herds located in the 2007 outbreak area would have been infected . This result indicates that production losses in non-reported infected herds could contribute to an important part of the total burden of the epizootic.Results found in this study are consistent with other publications. A follow-up survey of 50 dairy herds in Belgium conducted between December 2007 and February 2008 indicated that only 20 herds were officially notified as BTV outbreaks whereas all had been infected with BTV-8. The authors indicated that similar discrepancies occurred in all provinces Fertility could vary among regions due to differences in herd management and/or local weather conditions. The unexposed population considered in this study was composed of herds located in regions unexposed to the virus in 2007. A difference of fertility – independently of the exposure – between exposed and unexposed regions could bias the quantification of the effect of BTV-8 exposure. To check whether such a difference existed, the fertility before the beginning of BTV-8 epizootic, in 2005, was compared between the 2 regions exposed and unexposed. To do so, the same Cox model using the adjustment variables previously described was considered. No difference was shown, indicating that fertility was comparable before the exposure between the 2 regions. Therefore, the decreased fertility evidenced can be attributable to BTV-8 exposure and not to differences in herd management and/or local weather conditions. Finally, it was also check that there was no difference when comparing fertility between 2005 and 2007 in the exposed region.Coxiella burnetiiThe evidence of under-reporting of cases during an epizootic in the case of BTV-8 outbreak could be generalized to pathogens that affect cattle’s performance. In cattle, most pathogens, including zoonotic agents such as


Thermococcus kodakarensis (KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N′-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N′-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex.Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeon Cyclodextrins with six, seven, and eight glucopyranosyl moieties are termed as α-, β-, and γ-cyclodextrins, respectively. In water CDs adapt a structure with all hydrophilic groups directed towards exterior surface and hydrophobic groups towards an internal cavity. The internal hydrophobic cavity of CDs allows them to be resistant to hydrolysis by common amylases and also form inclusion complexes with different organic molecules [α-amylase superfamily), the largest family of glycoside hydrolases, is a member of clan GH-H along with families GH-70 and GH-77. The α-amylase family (GH13) is the most important family for industrial applications [Enzymatic hydrolysis of polysaccharides is a method of choice in many industrial processes due to its high efficiency and better yields of the products as compared to acid hydrolysis. Glycoside hydrolases have been used for processing of starch, cellulose, hemicellulose, and cyclodextrins . Cyclodeolecules , 3. CDs olecules , 4, 5. Tolecules . The famications . Althougications , 11.α-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), glucanotransferase (EC 2.4.1.25), and cyclodextrinase or cyclomaltodextrinase (EC 3.2.1.54). All of these subfamilies share a common catalytic domain comprising a TIM barrel or (β/α)8 barrel with a catalytic triad [β-strands only [ Thermococcus kodakarensis (KOD1) has also been reported to hydrolyze CDs into maltose or glucose [The family GH13 is further classified into 35 subfamilies with at least 26 different specificities , includiic triad  and a C-nds only . Many ennds only , 15. Thends only , 17. Rec glucose . The the glucose –21. Due  glucose , 23. Cur glucose . Till th in silico approach to provide insight into the sequence, structural components, domain arrangement, catalytic machinery, and enzyme-substrate interactions of thermophilic cyclodextrinase (Tk1770) from Thermococcus kodakarensis (KOD1), an enzyme of potential industrial applications. This study provides the first attempt to use in silico approach to provide insight into the structure and key components of catalytic machinery of cyclodextrinase (CDase) from T. kodakarensis.In this work we have used an T. kodakarensis KOD1  was retrieved from UniProtKB. A blast sequence similarity search was carried out against UniProtKB to find homologs of Tk1770. From the blast results sixteen different sequences of CD hydrolyzing enzymes from bacterial and archeal sources were selected for further studies (The amino acid sequence (UniProt ID Q5JJ59) of CDase from studies . The aliThe Tk1770 CDase was subjected to NCBI BLAST against RCSB PDB (Protein Data Bank) to search suitable template(s) for comparative modeling. Multiple X-ray crystallographic structures  with sequence identity from 56% to 29%, respectively, were selected as templates . The seqα-, β-, and γ-cyclodextrins) into the active pocket of Tk1770 was carried out using AutoDock and MGL Tools v1.5.6 [In order to investigate the enzyme-substrate interactions, the docking of substrates  from S. marinus is quite unique in this regard as it exhibits both CD hydrolyzing activity and additional N′-domain [A rooted phylogenetic tree was constructed from alignment using MrBayes with rate matrix wag (fixed) to find evolutionary relationship. The tree was divided into three clades with all bacterial enzymes forming one clade and archeal enzymes divided into two clades . The tre′-domain . It suggThe homology modeling program Modeller v9.14  was usedZ-score by comparing it with Z-score values of experimentally resolved protein structures in Protein Data Bank [In homology modeling, sometimes the model might contain certain high-energy loops or residues with unusual geometry. Thus, the model selected was refined using Modeller built-in loop-refinement function on loops ranging from 3 to 7 amino acids in length and then validated with ProSA-web server and PROCHECK analysis . The oveata Bank . Ramacha P. furiosus neopullulanase (PYRFU NPase) (PDB ID: 4AEF) for an analysis and comparison of the active site and other structural features. The overall structure of Tk1770 CDase folds into four major domains with two β-strands only N-terminal domains , connected to TIM barrel (A-domain) and a C-terminal domain, also consisting of β-strands. The structure of N′-domain of Tk1770 typically represents CBM48 with eight β-strands [ T. kodakarensis (85°C) and the optimum temperature for other archeal CD hydrolyzing enzymes [Homology model of Tk1770 CDase was aligned with-strands , 36. The enzymes .β/α)8 barrel (A-domain) also contains a much larger B-domain between β-strand 3 and alpha-helix 3 from residues 306 to 403. The B-domain of all the archeal enzymes possesses a helix-loop-helix (HLH) motif that extends at the entrance of active site  motif in the B-domain that is absent in all bacterial homologs. The homology model constructed revealed that loop connecting β-strand 7 and β-strand 8 of N′-domain extends into the catalytic site (A-domain) and plays an important role in substrate binding. Residues Y88, Y93, K94, F95, and R97 in extended loop of N′-domain of Tk1770 CDase are conserved in CD hydrolyzing enzymes of archaea. Structural alignment between model and template (4AEF) indicated that P91 and S92 in loop extension of N′-domain of Tk1770 might decrease its flexibility and interactions with A-domain. This might contribute to the decreased stability of two domains in Tk1770.Cyclodextrinase from hyperthermophilic archaeaThe docking studies indicated that residues K94, R97, K364, S375, D502, E504, and R550 form hydrogen bonds with substrates. Residue K364 in the helix of HLH motif extending at the entrance of the catalytic site interacts with substrate and might be involved in guiding the substrate into the catalytic site. From these results it can be inferred that archeal CD hydrolyzing enzymes have developed catalytic machinery in which an extension of N′-domain not only constitutes a part of active pocket, but also plays an important role in substrate binding.