To what extent are creative processes in one domain  affected by information from other domains ? While some studies of professional creators suggest that creative abilities are domain-specific, other studies suggest that creative avocations stimulate creativity. The latter is consistent with the predictions of the honing theory of creativity, according to which the iterative process culminating in a creative work is made possible by the self-organizing nature of a conceptual network, or worldview, and its innate holistic tendency to minimize inconsistency. As such, the creative process is not restricted to the creative domain; influences from domains other than that of the final product are predicted to impact the creative process and its outcome. To assess the prevalence of cross-domain influences on creativity we conducted two studies: one with creative experts, and one with undergraduate students from diverse academic backgrounds. Participants listed both their creative outputs, and the influences (sources of inspiration) associated with each of these outputs. In both studies, cross-domain influences on creativity were found to be widespread, and indeed more frequent than within-domain sources of inspiration. Thus, examination of the inputs to, rather than the outputs of, creative tasks supported the prediction of honing theory that cross-domain influences are a ubiquitous component of the creative process. Creative thought is central to human life. It shapes everyday activities such as putting together an outfit or holding a conversation. It fuels cultural evolution, giving us technology, music, media, and art. It fosters a sense of personal and cultural identity. Yet although it is perhaps our most defining human trait, it is one of the most elusive aspects of human cognition.problem domain . While the central aim of most theories of creativity is to account for the existence of creative products, arose to account for the cumulative, open-ended nature of cultural evolution. HT grew out of the view that humans possess two levels of complex, adaptive, self-organizing, evolving structure: an organismic level, and a psychological level . In short, HT posits that the worldview is the hub of a second evolutionary process—cultural evolution—that rides piggyback on the first—biological evolution—and that creative thinking fuels this second evolutionary process  versus cross-domain influences . We hypothesized that the majority of participants’ creative outputs will be inspired by cross-domain influences rather than within-domain influences.An internet search was conducted to locate individuals who were highly creative in any domain. They were recruited by email and invited to participate in the study on a voluntary basis. The method and recruitment email were both approved by the UBC Research Ethics Board. A total of 151 individuals participated.Participants were provided a link to an online questionnaire hosted by SurveyMonkey. The questionnaire asked their gender, age, and occupation, as well as the following open-ended questions:(1)What is the general category for the creative work for which you are most known ?(2)What is the subcategory for the creative work for which you are most known ?(3)Please describe your creative outputs.(4)Please describe as best you can your creative process.(5)Describe all elements that have inspired your work , and with each item, if possible, put as much identifying information as you can about the item it inspired . Do this for as many of your creative works as you can, itemizing them as (a), (b), (c), and so forth. Provide as much detail as possible.cross-domain (C), within-domain narrow (WN), within-domain broad (WB), and uncertain (U). A response was classified as WN if the domain of inspiration and the domain of the creative output fell within the same subcategory . An example of a WB influence is a painting inspired by a photograph. A photograph and a painting belong to the same domain of visual art, but not to the same sub-domain. Cross-domain influences were those for which the domain of the influence was different from the domain of creative expression. For example, a poem (domain: writing) that inspired a piano piece (domain: music) was rated as a cross-domain influence. An answer was categorized as U when insufficient information was provided to categorize the influence as WN, WB, or C. Examples of each category of influence in a few different domains of creative output are provided in Question four was not used in this analysis, as we chose to focus only on the inspirational elements provided. The influences provided in response to question five were divided into four categories: The rationale for using the self-report method was that only the participants themselves would be able to know what inspired their creative outputs. Outside assessments were not used in this study (or the next) since we were not interested in the quality of their creative contributions, nor in how creative other people assess these participants to be.SD = 2.29, Mdn = 3.00, range: 1–10).Of the 151 participants, 68 were excluded because they did not answer question five, which was the focus of our analysis. Five additional questionnaires were excluded because the raters were unable to understand the participants’ answers. This left a total of 78 participants  whose responses were analyzed. Nearly 58% of the participants were age 50 or older, and half of them worked in art/design. The participants provided a total of 298 influences, and the average number of influences provided by each participant was 3.82  = 45, p < 0.001. Due to the non-independence of the data, no significance testing was conducted for the differences in frequency for those who contributed two or more influences. Instead, we present the frequency of the occurrence of each category for our analysis.Since participants were encouraged to list all of the influences they could think of that inspired their creative outputs, there was variation in the number of influences that each participant provided. A possible explanation for the low frequency of WN influences (7.72%) could be that there is often only one WN influence that can match a given creative output, but there is a larger number of WB influences, and a potentially infinite number of C influences. Thus, once someone has provided an influence that matches the domain of the creative output, any subsequent influences they provide are more likely to be either cross-domain or within-domain broad. To explore the possibility that our results gave more weight to participants who reported multiple influences, we also analyzed the data according to the number of influences provided by each participant. The breakdown of data by the participants who gave the same number of influences  shows thThese results demonstrate that even if individuals primarily express their creativity in a single domain, they are often employing cross-domain thinking when they create. This study enriches our understanding of how the creative process works and adds to the growing body of evidence that creativity is much more than a matter of acquiring domain-specific expertise.The results of Study 1 prompted speculation as to whether or not the impact of cross-domain influences was limited to individuals with expertise in a particular creative domain. The goal of Study 2 was to assess whether the ability to develop creative works that employ cross-domain influences only comes online after the tools and techniques of a particular domain have been mastered, or whether it generalizes to individuals without expertise in a creative domain. Thus, the study investigated the hypothesis that regardless of one’s expertise in a creative domain, creative outputs will be inspired by cross-domain influences.The sample included 463 undergraduate students  from the University of British Columbia. They were recruited through SONA, an online participant pool approved by the UBC Research Ethics Board. Participants were granted one course credit  for their participation in this study.The SONA website provided a link to an online questionnaire hosted by FluidSurvey. The procedure and questions followed the same format as Study 1.SD = 1.92, Mdn = 3, range: 1–15). Nearly all participants (95.80%) were under the age of 30.Of the 463 participants, 111 were excluded because they left one or more of the written-response questions blank. (They were able to receive course credit whether or not they completed the questionnaire.) An additional 90 questionnaires were excluded because the participants did not provide classifiable answers to question five. Some of the questionnaires were unclassifiable because participants either provided a description of their creative process, or answered with what motivated a creative output, rather than providing an inspiration . The second category of unclassifiable questionnaires were those in which no creative outputs were provided in conjunction with a creative influence. The last category of unclassifiable questionnaires were those for which the raters found them incomprehensible and impossible to evaluate. This reduced the sample to 262 complete questionnaires for analysis , and these questionnaires provided a total of 758 influences. Each participant listed an average of 2.89 creative influences  The percentage of agreement between the two raters was 78.10%. Cohen’s kappa for two raters was moderate, κ = 0.50, but improved when partial weights were assigned to the WN/WB categories as in Study 1, κ = 0.56 . The ratA participant’s response to question 1 (their creative domain) was referenced alongside the types of output they recorded in question 5 in order to assign a general creative domain to each participant. We used nine categories in total. Participants who reported two or more outputs from different creative domains were categorized as ‘Multiple Domains.’ ‘Sport’ included dance and gymnastics, as well as team sports. ‘Art’ included sculpting, painting, and drawing. ‘Design’ included different types of design, such as fashion design, and interior design. ‘Other’ included those who did not fit into another category, such as ‘Law’ or ‘Administration.’The total number of influences in each category of influence, as well as the distribution among participants who gave the same number of influences, are provided in 2(3) = 64.59, p < 0.001.The results were highly consistent with those of Study 1. Of the 758 influences provided, 107 (14.12%) were WN, 81 (10.69%) were WB, and 564 (74.41%) were C. Thus, cross-domain influences constituted three times the number of within-domain influences, even when broad as well as narrow within-domain influences were included (24.80%). Of the 262 participants, 222 of them (84.73%) provided at least one cross-domain influence. Similar to Study 1, regardless of the number of influences provided, at least 64% of them were cross-domain. A chi-square test for the result of those who gave one influence showed that the difference in frequency of each category was significant, χOne possible reason for the high number of cross-domain influences for artists and writers is that in these domains creative expression is highly valued, while works that are closely related to previous books/paintings may be derided for their lack of creativity. Moreover, individuals may be specifically drawn to these domains because this kind of expression is accepted and even encouraged. In other domains, it may be more important to draw upon the ideas and methods of previous creators, resulting in more within-domain influences. Cross-domain influences for ‘Sport’ in our sample may be due to dancing being included in this category, and common influences for dance included music, lyrics, and emotions . Future studies with more complete and representative samples of different creative domains may be able to examine the distinctions within these domains more closely .The results of this study concur with those of Study 1 in that the majority of creative outputs were inspired by cross-domain influences. In addition, Study 2 shows that this is not only the case for individuals with proven success or expertise in a creative domain, but holds true for non-experts as well. Except for WN, the confidence intervals from the two studies overlap, indicating that there is no significant difference between the results of Study 1 and Study 2 see .This result has implications for our understanding of the creative process, because it demonstrates that it is substantially less domain-specific than it is widely presumed to be. Even if individuals primarily express their creativity in a single domain, they are often employing cross-domain thinking when they create. Although domain-specific knowledge may ensure that tools of the trade are appropriately applied, and one’s creative works may consistently be in one particular domain, the sources that initially triggered these creative processes may be diverse in nature. To the extent that this is the case, creativity may involve synthesizing information from different arenas of one’s life. This is consistent with high levels of functional connectivity amongst the default, salience, and executive neural circuits in creative individuals during creative thinking . AlthougAt first glance it might seem that the basic units of cultural evolution  are such things as catchy songs, rituals, or tools. However, the above evidence for the cross-fertilization of different domains suggests that the only way to delineate the cultural lineage of a given idea is to look to the creator’s entire loosely integrated web of knowledge and understandings . In this way, the inspirational sources—or ‘conceptual parents’—of a sad ballad could include everything from other musicians, to the patter of rain, to the death of a loved one. Thus, creative products don’t just serve practical purposes or provide aesthetic pleasure; they provide tangible external markers of the evolutionary states of the worldviews that generated them. This is consistent with the theory that what evolves through culture is, not creative outputs like songs or tools, but worldviews, with creative outputs as the externally visible ‘excrement’ of this transformation process , 2013. IAs discussed elsewhere, a worldview not only self-organizes in response to perturbations but it is imperfectly reconstituted and passed down through culture . This isWe attribute the large fraction of respondents who did not complete the questionnaire to the anonymous nature of the study. In future studies, it would be helpful to consider ways of further incentivizing participants to complete the questionnaire. Although the open-ended nature of the questionnaire was necessary to enable participants to provide anything as an inspirational source, it may have been less inviting for those who prefer a structured format. Changing the format from an online questionnaire to an in-person interview may elicit a higher response rate. In addition, the instructions should actively discourage participants from providing motives  instead of inspirational sources, and examples of each should be given so that the distinction is clear. Also, reformatting the questionnaire so that it is always clear which creative influence is associated with which creative output would help clarify the interpretation of the responses.A limitation of the self-report format is that participants might not accurately remember, or report the inspirations for their creative works. Related to this is that the open-ended nature of participants’ responses meant that they varied in detail and in the extent to which they actually responded to the particular question that was asked. However, we believe the methods chosen matched the goals for this study. There is no easy or standardized way of examining the creative influences of others. Our goal was to obtain the most information from each participant possible by allowing them to respond in their own words, with as much detail as possible, rather than to limit their responses by a more formalized structure such as a survey. Our study showed that when people self-report their creative inspirations, cross-domain influences are frequently reported. Besides the above-mentioned ideas, other possible future methods could include the identification of a creative output, and then to approach the creator for an explanation of their influences for that particular output. This study also made no effort to measure or control for the creativity of the outputs, but this variable could be explored in future studies, to assess its relation to the domains of influence. Another suggestion for future work would be to add a second, within-domain example to the instructions, such as “My Sonata was influenced by my recent obsession with Beethoven’s Pathetique.”We note as another limitation that the gender balance was skewed toward females, and Study 1 participants tended to be older than Study 2 participants. The age difference arose largely from the difficulty of finding accomplished creative professionals under age 30. In future studies it would be advisable to endeavor to obtain a more equal gender balance and age distribution to ensure that these factors do not affect the results.An important direction for future research is to investigate the timing and underlying mechanisms of cross-domain influence. In the studies reported here, participants were prompted to focus on the inspiration for their creative outputs, but we note that cross-domain influences are not necessarily exclusive to the inspiration phase of the creative process. To better understand the frequency and timing of cross-domain thinking in the creative process, further research needs to be done on other phases of the creative process, and the process as a whole.In regards to the mechanisms of cross-domain influence, Another direction is to investigate the hypothesis that individuals in some creative domains make less use of cross-domain influences than others , and explore the effect of different types of influence on the level of creative output within that domain. For example, within-domain influences may be important for a field such as science, where domain-specific knowledge is essential , but croThe finding that the majority of creative outputs were inspired by cross-domain influences has implications for the development of practices that promote creativity in education, the workplace, and personal life. There is an international trend toward streamlining classwork and content to the basics, and reducing or eliminating classes in the arts. Educational systems are increasingly geared toward providing much in the way of information but little in the way of examples of, or opportunities to cultivate, creative thinking skills. The results of this study suggest that creativity may be cultivated by interdisciplinary courses, as well as activities that foster connections between different domains, such as painting murals of ecological food chains or writing poetry about science, as well as exposure to such cross-disciplinary cultural outputs.The method and recruitment email were both approved by the UBC Research Ethics Board at the University of British Columbia (Okanagan Campus). Written consent was obtained from all participants.LG designed the research. NC conducted Study 1. SW conducted Study 2. VS analyzed the studies. VS and LG wrote the manuscript. All authors contributed to the manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, the formability of woven carbon-fiber (CF)-reinforced polyether-ether-ketone (PEEK) composite sheets in the solid-state thermoforming process were investigated, and the failure mechanisms were discussed. The formability of the woven CF/PEEK sheets were analyzed using flexural tests, Erichsen test, and microscopic observation. The results show that the formability of CF/PEEK sheets significantly increases as the temperature rises from 165 to 325 °C, and slightly decreases as the deformation speed rises from 2 to 120 mm/min. The deformation of the sheets is caused by plastic deformation, shear deformation and squeeze deformation, without plastic thinning and fiber slippage, which is due to the restriction of the solid matrix and locked fibers. Moreover, the wrinkles will cause fiber fracture at lower temperatures and delamination at higher temperatures. At higher temperatures, the wrinkles mainly occur at the position with [0°/90°] fibers due to the squeezing of the matrix and fibers. In the last three decades carbon fiber (CF)-reinforced polymer composites (CFRP) have made their way into the aerospace and automotive industries as structural elements because of their high specific stiffness and strength ,2,3. Recg) and the melting point (Tm)  fibers due to the squeezing of the matrix and fibers, and few occur in the position with [−45°/45°] fibers.


Cyclic deformation at low-stress amplitudes of a rare earth-containing magnesium alloy (Mg–Gd–Y–Zr) was investigated with emphasis on the responsible microstructural relationship between deformation mechanism and fatigue crack initiation. The results show that the microstructural deformation is extremely inhomogeneous at the low-stress amplitudes. Both deformation twinning and non-basal slip are barely observed, and basal slip is the predominant deformation to accommodate micro-plasticity. Fatigue crack initiation occurred at the basal slip bands, causing the morphology of facet on the fracture surface. Therefore, the basal slip is of prime importance in low-stress cyclic deformation and fatigue failure, and fatigue improvement could potentially be obtained through hindering the motion of basal dislocation by microstructural obstacles. Magnesium alloy is an attractive structural material that can be potentially applied in automobile and aerospace industries, due to its low density. However, its strength is relatively low as compared with aluminum alloys. The addition of rare-elements (RE) in Mg alloys could improve the strength remarkably and tailor the deformation mechanisms . The strIt was known that a limited number of independent slip system is available in hexagonal close-packed (hcp) structure at room temperature. The deformation of Mg alloy is generally accompanied by dislocation slip and twinning ,3. As coThis work aims at investigating the cyclic deformation mechanisms at low-stress amplitudes responsible for Mg–RE alloy and unraveling its relation to the fatigue crack initiation mechanisms. The research results of this study may provide a deep understanding of high cycle fatigue failure in rare earth-containing Mg alloys.The material used in this study was an extruded magnesium alloy with a diameter of 10 mm. The chemical composition (wt %) is 10Gd, 3Y, 0.5Zr and Mg in balance. The tensile test  was conducted at room temperature according to the GBT228.1-2010 test standard (Chinese Standard Method). The yield and ultimate strengths of this alloy are 203.8 MPa and 258.1 MPa, respectively. The elongation at the final fracture is 11.5%. In fatigue testing  dimensions of the specimen are presented in It should be noted that the selected zone of EBSD analysis is about 1.0 mm away from the fracture surface, so the obtained results were caused by cyclic deformation instead of crack propagation-induced deformation near the fracture surface.5 cycles). The ratio of the cyclic number (Nc) to total fatigue life (Nf) is inserted within each image. The grain surface became a bit rough almost from the beginning of fatigue test (1.16%), due to the activation of basal slip. Along with the accumulation of micro-plasticity, the grain was filled with slip markings. Finally, a crack initiated from the slip bands as indicated in In order to obtain the crystallographic orientation of the crack initiation site, an area containing secondary cracks on the surface of the specimen at 130 MPa was analyzed by EBSD, as shown in After the crack initiation along slip bands, it would propagate from the surface into the matrix. To gain further understandings of crack behaviors beneath the surface, focus ion beam (FIB) technique was used to section the slip-induced crack. The final fracture surface was observed by SEM to investigate fatigue failure mechanisms. At low-stress amplitudes, the fatigue limit of Mg–Gd–Y alloy is in the range of 100~120 MPa ,7,23, inAt the stress amplitudes below 130 MPa, both twinning and non-basal slip were barely activated. Plastic deformation is mainly accommodated by basal slip. This could be ascribed to increased critical resolved shear stress in polycrystalline Mg alloys;Low-stress deformation in Mg alloy was extremely inhomogeneous, due to its limited slip systems. Slip markings were restricted within isolated grains that have favorable orientation for the basal slip;Fatigue cracks always initiated along surface slip bands, and then propagated into the matrix straightly along basal plane, resulting in facet morphologies at fracture surface;Since basal slip was responsible for predominant deformation and fatigue crack initiation, introducing of specific microstructure or interfaces that are capable of hindering <a> dislocation may significantly improve high cycle fatigue strength.Cyclic deformation mechanism of a rare earth-containing Mg alloy at low-stress amplitudes was investigated for further understanding of high cycle fatigue crack initiation. The following main conclusions can be drawn:


Salmonella hook, which has been studied most over the years, has not been solved at a high enough resolution to allow building an atomic model of entire FlgE for understanding the mechanisms of self-assembly, stability and the universal joint function. Here we report the structure of Salmonella polyhook at 4.1 Å resolution by electron cryomicroscopy and helical image analysis. The density map clearly revealed folding of the entire FlgE chain forming the three domains D0, D1 and D2 and allowed us to build an atomic model. The model includes domain Dc with a long β-hairpin structure that connects domains D0 and D1 and contributes to the structural stability of the hook while allowing the flexible bending of the hook as a molecular universal joint.The bacterial flagellum is a motility organelle consisting of a rotary motor and a long helical filament as a propeller. The flagellar hook is a flexible universal joint that transmits motor torque to the filament in its various orientations that change dynamically between swimming and tumbling of the cell upon switching the motor rotation for chemotaxis. Although the structures of the hook and hook protein FlgE from different bacterial species have been studied, the structure of  The bacterial flagellum is a motility organelle and is a complex nanomachine consisting of more than 20 different proteins in different copy numbers ranging from a few to a few tens of thousands . BacteriSalmonella is powered by proton motive force across the cell membrane and rotates the filament at around 300 revolutions per second [Salmonella has several peritrichous flagella that form a bundle behind the cell to produce strong thrust, but the switching of motor rotation to clockwise direction causes a polymorphic transition of the filament to a few different right-handed supercoils, thereby making the bundle to fall apart, causing cell tumbling for chemotaxis and thermotaxis [The rotary motor of r second ,6,7. Ther second , which ir second ,9. The frmotaxis ,11,12. TSalmonella hook is a short, highly curved tubular structure built by helical assembly of about 120 copies of a single protein, FlgE [The in, FlgE ,14,15,16in, FlgE , and itsin, FlgE ,19,20. The distal end of the hook is connected to the filament via two hook-associated proteins, FlgK and FlgL, while its proximal end is directly connected with the rod within the basal body. The rod is rigid as a drive shaft that transmits motor torque to the filament through the hook. The rod, hook and filament form the axial structure of the flagellum and show structural similarity to each other as tubular structures composed of 11 protofilaments. Nevertheless, their diameters, mechanical properties and functions are distinct from each other. The rod consists of four different proteins, FlgB, FlgC, FlgF, and FlgG  in diffeSalmonella enterica serovar Typhimurium by X-ray crystallography and electron microscopy (cryoEM) for many different parts [Salmonella flagellum [Campylobacter jejuni revealed the domain structures that are nearly identical to those of Salmonella hook FlgE and also resolved the structure of domain Dc clearly as an L-shaped, long β-hairpin named L-stretch [Salmonella hook, but the length of the Salmonella peptide chain assigned to this Dc domain is much shorter than that of Campylobacter by 17 residues. So a question still remains as to how domain Dc with the β-hairpin conformation interacts with neighboring FlgE subunits to stabilize the entire hook structure without reducing its bending flexibility. The structure of the bacterial flagellum has been intensively studied for nt parts . The crylagellum ,26,27, alagellum ,26,27. Tlagellum . A recen-stretch . This loSalmonella hook to build its complete atomic model. With recent advances in cryoEM techniques with the use of a direct electron detector camera for high-resolution image data collection, we have obtained a 3D density map at 4.1 Å resolution and built an atomic model that reveals the conformation of domain Dc and its intimate interactions with neighboring subunits. We describe the nature of these intersubunit interactions and discuss the role of domain Dc in the structural stability and universal joint function of the hook.We therefore used cryoEM image analysis to obtain a high-resolution structure of Salmonella enterica serovar Typhimurium (ΔfliK mutant) according to the protocol by Aizawa et al. with slight modifications [2, and cryoEM images were recorded in the Super-Resolution mode of K2 camera with an image size of 7676 × 7420 pixels, at a nominal magnification of 50,000×, a defocus range of 0.5–2.5 µm, and a dose rate of 2.2 electrons/pixel/sec. Images were recorded in a dose fractionation mode, with a total exposure time of 6 s at a frame rate of 0.2 s/frame, and so 30 frames were recorded for each movie image, with a total cumulative dose of 82 electrons/Å2.Flagellar polyhooks were isolated from the SJW880 strain of ications . After iBeam-induced sample motions on dose-fractionated frames were corrected by GPU accelerated MotionCor2 software . First gIn total, 42,293 segment images were extracted from 486 micrographs, 2D classification was performed, and 41,260 segment images were used for further image processing. In the 3D classification step, local search of helical symmetry  was performed with initial parameters of 64.7° for the twist angle and 4.1 Å for the axial rise . In the Salmonella hook (PDB ID: 3A69) [Salmonella FlgE domain Dc, and the 3D model template was the corresponding domain Dc of C. jejuni FlgE (PDB ID: 5JXL) [The previous atomic model of D: 3A69)  with an D: 3A69) . The tarD: 5JXL) . The seqD: 5JXL)  and refiD: 5JXL)  showed tSalmonella FlgG including domain Dc was also built by MODELLER [Salmonella FlgG, and the 3D model template was again the corresponding Dc domain of C. jejuni FlgE (PDB ID: 5JXL) [Salmonella FlgG (PDB ID:5WRH) [A homology model of MODELLER  using a MODELLER  as a temD: 5JXL)  as well ID:5WRH) . The RamID:5WRH)  by a modID:5WRH) . https://www.ebi.ac.uk/pdbe/emdb/) under accession code EMD-9974, and the atomic model coordinates have been deposited to the Protein Data Bank (https://pdbj.org) under accession code 6KFK.The cryoEM 3D density map has been deposited to the Electron Microscopy Data Bank  form continuous helical arrays along the 6-start helical lines on the hook surface but have no interactions along −5-start and 11-start helices, and therefore, these six stranded helical arrays of D2 domains are widely apart from each other. The D1 domains (blue) form a mesh-like array along all three major helical lines but their interactions are not very tight with a small gap in all the directions, suggesting that these interactions modestly contribute to the stability of the hook structure. The D1 domains are probably interconnected indirectly through one or two layers of water molecules between their domain surfaces, although water molecules are not visible at this resolution . These lIn contrast, the Dc domains (green) have close interactions in the −5-start and 11-start helical lines, making a stable tubular structure, albeit the 6-start interactions are absent. The D0 domains (red) form intimate interactions in all the directions, with the N- and C-terminal helices forming a two-layer helical bundle where the C-terminal helices tile the inner surface of the central channel of the hook with polar and charged residues. The N-terminal helices, not facing the channel, have interactions only in the −5-start helical line, but together with Dc domains, they are involved in intricate intersubunit interactions. As described in the previous section, the N-terminal helix of domain D0 and the long β-hairpin of domain Dc form a tight interaction, strongly suggesting that domains D0 and Dc are likely to behave as one domain D0-Dc. A stretched loop of subunit −11 formed by residues 64–67 at the top of domain Dc connecting to domain D1 is involved in close tripartite interactions with the tip of the long β-hairpin of domain Dc (residues 40–52) and the N-terminal end (residues 1–7) of subunit 0  of a short β-hairpin of domain D1 of subunit −5  is still present to allow the compression of the protofilament for flexible bending of the hook (Salmonella FlgG and FlgE share a highly homologous sequence with 32% identity by BLAST search [The flagellar rod is a drive shaft of the rotary motor, transmitting motor torque to the filament acting as a helical propeller through the hook as a universal joint. The rod is a helical tubular assembly of four proteins, FlgB, FlgC, FlgF, and FlgG, but its major part rotating inside the LP ring of the flagellar basal body is the distal part of the rod formed by FlgG . The diaT search , except C. jejuni also has a similar sequence to Salmonella FlgG, with 17 extra residues in this region compared with Salmonella FlgE (Campylobacter FlgE than Salmonella FlgE as well. In agreement with this prediction, the recent cryoEM structure of the hook isolated from C. jejuni showed an L-shaped, long β-hairpin named L-stretch in domain Dc of FlgE [Salmonella FlgE. So we used this structure to build a homology model of the β-hairpin of Salmonella FlgG and inserted it into the atomic model of FlgG in the polyrod solved by cryoEM analysis [On the other hand, FlgG has a region of 18 extra residues in the domain Dc region, suggesting that the β-hairpin of domain Dc is longer than that of FlgE a. FlgE olla FlgE b, sugges of FlgE , which ianalysis  in orderSalmonella FlgE in blue, Campylobacter FlgE in red and Salmonella FlgG in green. The relative disposition of the β-hairpin to the N- and C-terminal helices of domain D0 is nearly the same between Salmonella FlgE and Campylobacter FlgE (Salmonella FlgG and Campylobacter FlgE (Salmonella FlgG and FlgE (Salmonella hook, Salmonella rod and Campylobacter hook, as presented in Salmonella FlgG and Campylobacter FlgE are projecting out into the outer layer of their tubular structures composed of the D1 domains and are filling the gap formed in the helical array of the D1 domains to further stabilize and rigidify the tubular structure. Pairwise comparisons by superposing the D0 domains are shown in ter FlgE  left andter FlgE  middle aand FlgE  right, band FlgE  middle. Campylobacter hook is curved in the native form and functions as a universal joint, it easily becomes straight and appears to be more rigid than Salmonella hook [Campylobacter hook needs to be more rigid than Salmonella hook, which is maybe because Campylobacter has two polar flagella and the hook needs to be rigid in order to keep orienting the flagellar filaments in the axis of the cell body to produce a thrust efficiently. The insertion of extra 18 residues of the Salmonella FlgG-specific sequence into Salmonella FlgE was expected to make Salmonella hook as rigid as Salmonella rod. The hook formed by this insertion mutant of FlgE actually became straight and rigid just like the rod, and the flagellar filaments on these mutant Salmonella cells were all spread apart and could not form a bundle to produce thrust for swimming even though individual motors were rotating rapidly [Although lla hook , and thiThus, the long β-hairpin of domain Dc plays an important role in the flagellar rod and hook structure, regulating and optimizing the structural stability and rigidity of the entire tubular assembly by changing its length for their required functions. The cryoEM structures of the flagellar rod and hook clearly revealed it, demonstrating the power of the cryoEM technique as a unique and essential tool for structural biology capable of visualizing native, functional structures of biological macromolecular assemblies. Salmonella in the straight form by cryoEM helical image analysis and built an atomic model based on the 4.1 Å resolution map. This model revealed the entire structure of the hook component protein FlgE consisting of domains D0, D1 and D2 as well as the long β-hairpin connecting domains D0 and D1, which is involved in intricate intermolecular interactions to stabilize the tubular structure of the hook while maintaining its bending flexibility. This structural feature is distinct from that of the flagellar rod consisting of FlgG with a much longer β-hairpin, which further stabilizes and rigidifies the rod structure to work as the drive shaft of the flagellar motor. We carried out the structural analysis of the flagellar hook isolated from


In this microreview, we would like to reflect on the ideas put forward, with special focus on protein domain architecture of the enzyme involved, its orthologues in plants, and the implications of the differential effects observed between binding of the two alarmone molecules, ppGpp  and pppGpp (guanosine-5′-triphosphate-3′-diphosphate). In our previous work, we showed that the Escherichia coli nucleotide 5'-monophosphate nucleosidase, PpnN, which is conserved in Proteobacteria, cleaves its preferred substrate, guanosine monophosphate (GMP), at a much higher rate in the presence of both pppGpp and ppGpp  increased bacterial sensitivity towards antibiotics. In contrast, complete loss of the ppnN gene has the inverse effect, i.e. reduced competitive growth and improved antibiotic tolerance. We used these observations to propose a model in which E. coli uses PpnN to balance the need of fitness (fast growth) against tolerance towards antibiotics to improve survival.In our recent publication (Zhang nd ppGpp . Structu Finally, we show using sequence alignments that the domain structure of PpnN and thus function of the DUF domains is widely conserved across many Proteobacteria. With these new insights in mind, we suggest that DUF4478 and DUF3412 be re-named as (p)ppGpp-Activated Gating (PAG) domains 1 and 2 (PAG1 and PAG2), respectively. A search for similar domains in the PFAM database reveals that in the majority of cases, two PAG domains flank a nucleosidase-like domain as observed in PpnN. In a few cases, however, shorter versions of the enzyme are found in which either one PAG domain or the catalytic core plus a PAG domain is missing. Structural analysis suggests that a catalytic core plus a single N-terminal PAG1 domain could be sufficient for oligomerisation (tetramerisation) and thus may support cooperative substrate binding even in the absence of regulation by (p)ppGpp. Alternatively, it is also possible that the truncated proteins represent non-functional PpnN orthologues, but this hypothesis awaits experimental confirmation. Our data show, however, that loss of a functional PpnN enzyme leads to slow growth and render bacteria more tolerant towards antibiotics, a proposed step towards developing resistance. This idea is supported by the observation that several of the truncated ppnN genes occur in pathogenic, multi-drug resistant bacteria, some of which have been isolated from patients.PpnN consists of three domains, a catalytic monophosphate nucleosidase domain (MND) with a Rossmann-like fold, and two Domains of Unknown Function (DUFs) in the N and C termini of the protein, DUF4478 and DUF3412, respectively. The enzyme forms a tetramer via interactions between the N-terminal DUF4478 domain and the neighbouring MND. Moreover, residues from both the N and C-terminal DUF domains come together to form binding pockets for pppGpp between neighbouring monomers. In this way, the DUF domains control access to the active site by structurally occluding it in the .While searching for orthologues of PpnN, we came across a protein family in plants, the so-called LOG ("lonely guy") proteins, which are responsible for the activation of the phytohormone cytokinin through cleavage of the N-glycosidic bond. This reaction is chemically highly similar to the one carried out by PpnN Arabidopsis thaliana, LOG3 and LOG8, both of which form a symmetrical dimer via interactions near the conserved PGGxGTxxE motif, which is predicted to be involved in catalysis. The LOG protein monomer structures are highly homologous to the central MND of PpnN . Intriguingly, when superimposing one monomer of a LOG dimer onto a PpnN monomer, it becomes evident that there are conserved structural elements between the PAG2 domain and the C-terminal region of the other LOG monomer . The similarities are found close to the MND of PpnN and cover several structural elements in the C-terminal PAG2 domain that act as a gate to the active site in PpnN. This suggests that the dimerisation of LOG could function to regulate activity in a similar way to the PAG domains in PpnN. It thus appears that there could be an evolutionary connection between PpnN and LOG proteins in which both evolved from a primordial, simpler enzyme consisting only of a single catalytic domain. A gene duplication event could then have led to the presence of one or more PAG domains. Despite the similarities, the two protein families differ and we have found several bacterial PpnN orthologues wrongly annotated as LOG proteins in available databases, which should be revised. We suggest that genes encoding all three domains, PAG1, MND, and PAG2 should be annotated as a ppnN orthologue.There are two available structures of LOG proteins from E. coli). In our study, we tested the catalytic rate of PpnN versus the concentration of the substrate (GMP) in the absence of alarmone or presence of a constant concentration of either ppGpp or pppGpp. When comparing the activity at the physiological substrate concentration, addition of ppGpp only slightly increases the activity of PpnN compared to the situation where no alarmone is present, but surprisingly appears to affect the cooperativity of substrate binding based on the calculated Hill coefficient. Conversely, binding of pppGpp appears to maintain cooperative substrate binding while increasing the activity dramatically. Although a more thorough analysis is required to uncover the kinetics of ppGpp and pppGpp binding to PpnN, as well as how binding affects the subtrate binding, these preliminary data are consistent with what has been found for the Bacillus subtilis small alarmone synthetase (SAS), RelQ. In this Gram-positive organism, the alarmone synthesis activity of RelQ is allosterically stimulated by pppGpp, but not ppGpp, suggesting that ppGpp and pppGpp, despite having very similar chemical structures, indeed show differential effects. Furthermore, intracellular concentrations of ppGpp and pppGpp are known to be very dynamic in E. coli, ranging from 40 µM during exponential growth, to 810 µM upon transition to stationary phase, and 150 µM in stationary phase, respectively. Additionally, a preferential production of either ppGpp or pppGpp is observed in various strains when exposed to different types of stress. In E. coli, the enzyme GppA is known to quickly convert pppGpp to ppGpp. Finally, other ppGpp analogues such as pGpp, ppGp, or pGp have recently been discovered by both us and other groups as well. Whether and how these molecules affect the activity of PpnN is currently unknown. Moreover, we know little about how the activity of PpnN is regulated temporally during bacterial adaptation to stress. Similarly, it is unclear how the regulation of PpnN in particular, and other (p)ppGpp targets in general, contribute to bacterial fitness. These questions remain important to answer, and are currently under study in our laboratories.Another interesting aspect not fully discussed in our previous publication is the large shift in activity and cooperativity of PpnN when bound to ppGpp versus pppGpp, especially at the physiologically relevant level of GMP  protein domains. Some LOG proteins, and some PpnN orthologs, have been wrongly annotated as lysine decarboxylases. Studies have already shown that the LOG proteins rather function as nucleosidases and not lysine decarboxylases, and this argument can be extended to PpnN. With the new data in hand we propose a domain nomenclature in which PpnN-type enzymes consist of the newly identified PAG1 (formerly DUF4478), MND (based on its Monophosphate Nucleosidase Domain activity), and PAG2 (formerly DUF3412) domains.Recently published data on the bacterial nucleosidase PpnN has helped clarifying how in vivo and in vitro means that we can start to answer more complex questions as above with regards to the stringent response. We already have data that shows that PpnN has a different response to two very similar molecules, ppGpp and pppGpp, indicating that the regulation is more complex than an ON/OFF switch. Testing the activity of proteins at alarmone pool levels resembling natural growth states could help us gain a greater insight into how bacteria regulate their growth, both with and without stress.Constant improvements in the limits and methods for detecting alarmones, both


Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization  was examined using confocal microscopy. While the two formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay—a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant  The eukaryotic cytoskeleton represents a dynamic network of protein filaments and tubules that has been extensively studied in a variety of model systems. In addition to basic cellular functions, including nuclear division, cytokinesis, organelle positioning, membrane trafficking and cell expansion, the cytoskeleton plays an important role in processes such as polar cell growth, cell division plane positioning, or cell to cell communication, which are essential for proper morphogenesis and thus the development of multicellular bodies in both metazoans  and planFormins, or FH2 proteins, members of an evolutionarily ancient and widely expressed family of eukaryotic cytoskeletal organizers, are a prominent example of such conserved components of the cell’s morphogenetic machinery. They are generally characterized by the presence of the conserved formin homology 2 (FH2) domain whose dimer can nucleate and cap actin filaments. In addition to other variable regulatory domains, the FH2 domain is usually accompanied by the proline-rich formin homology 1 (FH1) domain that can bind actin-profilin complexes, providing a substrate for formin-driven assembly of unbranched actin filaments . HoweverArabidopsis thaliana genome harbors 21 formin-encoding loci that can be divided into two phylogenetically distinct classes present in all angiosperms, referred to as Class I and Class II, based on their sequence similarity and domain organization [Physcomitrella patens For2A formin whose PTEN-like domain interacts with PIP2; this interaction is necessary for formin localization to the tip of apically growing cells [The nization ,17,18. Mng cells . Similarng cells  and inteng cells  and whicng cells .A. thaliana Class II formin, AtFH13 (At5g58160) that shares overall domain structure composition with AtFH14, using in planta transient heterologous expression in native Australian tobacco (Nicotiana benthamiana) leaves. While both formins can associate with microtubules (MTs) and the endoplasmic reticulum (ER), they exhibit distinct subcellular localization patterns. In addition, we demonstrate that although these formins show different pattern of localization, their FH2 domains are capable of heterodimerization—a first such observation in plant formins.In this study, we compare the intracellular localization of AtFH14 with that of a hitherto uncharacterized 2+-binding domain from protein kinase C), the proline-rich FH1 domain and the C-terminal FH2 domain. Although they share the overall domain structure, AtFH13 and AtFH14 are only 52% identical in their PTEN-like and C2 domains and 60% identical in their FH2 domains, and represent branches of the Class II formin clade that are clearly separate at least within Brassicaceae (Both AtFH13 (At5g58160) and AtFH14 At1g31810) are typical Class II formins [10 are tysicaceae a, and posicaceae b.N. benthamiana leaves. To examine contribution of individual domains of both proteins to their localization, we also prepared constructs expressing YFP-tagged N-terminal fragments of either protein containing the PTEN-like and C2 domains, as well as green fluorescent protein (GFP) and YFP-tagged isolated FH2 domains of AtFH13 and AtFH14. In addition, we also generated an YFP fusion of the N-terminal (PTEN-like and C2 domain-containing) fragment from a fortuitously cloned AtFH13 variant missing 21 amino acids within the PTEN-like domain (denoted PTENΔ) as a putative inactive variant of the PTEN domain  or a previously characterized variant of AtFH14  tagged bN domain c.N. benthamiana leaf epidermis transformation, individual transformed cells exhibited variable signal intensity. Confocal imaging of YFP-tagged AtFH13 in cells with a relatively weak signal showed fibers, suggesting cytoskeletal association, as well as punctate structures of varying size in the cortical cytoplasm -RFP, respectively. Neither AtFH13 nor AtFH14 showed association with actin fibers  but bothTo confirm functional connection between the AtFH13-labelled foci and the microtubule cytoskeleton, we examined the effect of the microtubule depolymerizing drug oryzalin on localization of AtFH13-YFP co-expressed with KMD-RFP. The oryzalin treatment caused partial redistribution of the cytoplasmic dots and increased their mobility, which may be due to cytoplasmic streaming, not affected by microtubule disruption . This suSince both AtFH13 and AtFH14 contain the PTEN-like domain known to associate with membranes ,24, we hNext, we compared the localization of isolated FH2 domains of AtFH13 and AtFH14 in transiently transformed epidermal cells. GFP-tagged FH2 domain of AtFH13 was predominantly cytoplasmic with a few small punctate structures; in contrast, YFP-tagged FH2 domain of AtFH14 exhibited prominent fibrous distribution similar to the full-length protein a. IntereTo assess the possible contribution of the membrane-binding PTEN-like domain to the observed peripheral association of AtFH13 and AtFH14 with the ER, we examined the subcellular localization of YFP-tagged PTEN-like domains of AtFH13 and AtFH14. For control, we also included a fortuitously cloned deletion derivative of the PTEN-like domain from AtFH13, PTENΔ_FH13, presumed to be inactive due to disruption of a structurally important segment of the protein. The YFP-tagged PTEN domain of AtFH13 accumulated in structures reminiscent of the ER in the cytoplasm, whereas an analogous derivative of AtFH14 decorated small cytoplasmic punctate structures while exhibiting also strong accumulation in the nucleus and weak cytoplasmic background. In contrast, the deletion derivative of the AtFH13 PTEN-like domain showed only diffuse cytoplasmic distribution, consistent with the deletion altering the protein´s folding and consequently its subcellular localization a. Co-expIt is well established that formins can form homodimers through their FH2 domains  but therIn the yeast two hybrid system, an isolated FH2 domain of AtFH13 can form homodimers as expected. However, in our two-hybrid setup the FH2 domain of AtFH13 also readily formed heterodimers with the FH2 domain of AtFH14 . This isPhyscomitrella patens [Formins are an evolutionarily ancient family of proteins ,11,12 tha patens ,24 and ia patens . Thus, ta patens ,29). In a patens ,33,34,35in planta expression system to document in vivo subcellular localization of fluorescent protein-tagged derivatives of two Arabidopsis Class II formins, the hitherto uncharacterized AtFH13 and somewhat characterized AtFH14 (see below). Although protein tagging by fusion with a relatively large polypeptide such as the YFP or GFP might influence its intracellular localization, such fusion proteins, including a plant formin [In this study, we use a heterologous t formin , have bet formin . Anothert formin , with tht formin .Both formins studied exhibit the typical domain composition involving a N-terminal PTEN-like domain followed by a repetitive, proline-rich FH1 domain believed to mediate profilin-actin binding and by the C-terminal conserved dimerizing and actin-nucleating FH2 domain ,18. Despin planta system co-expression of YFP-tagged full length AtFH14 or AtFH13 with actin markers did not reveal co-localization with the actin cytoskeleton, consistent with the strength, or indeed presence, of formin-microfilament association depending on the cellular context and molecular environment. Similar behavior has also been observed for the Class I formin AtFH4 which can bind and nucleate actin in vitro but does not decorate actin filaments in vivo [Formins, as known actin nucleators, can be expected to associate with actin filaments. Indeed, although AtFH14 was shown to preferentially bind microtubules, some co-localization of AtFH14 and actin was reported upon heterologous expression in mitotic tobacco BY2 cells . In addi in vivo . Surprisin vivo, and documented microtubule co-localization also for AtFH13. Remarkably, AtFH13 decorated the microtubules in a noticeably more discontinuous manner than AtFH14, often forming distinct particles along the microtubules. Mobility of these particles was greatly increased by the MT-depolymerizing drug oryzalin, proving that their organization is microtubule-dependent. Moreover, while the isolated FH2 domain of AtFH14 readily localized to the microtubules, the FH2 domain of AtFH13 was predominantly cytoplasmic, clearly indicating functional differences between the two FH2 domains. Analogously, metazoan formins have been documented to vary in their abilities to interact with microtubules and showed distinct localization patterns [Several Class II plant formins including AtFH14 were shown to interact with microtubules, sometimes more readily than with actin filaments ,23,39,40patterns ,42.Interestingly, while both AtFH13 and AtFH14 associated with microtubules, only in the later the FH2 domain, which is also responsible for actin binding, was sufficient for microtubule interaction. Direct binding of FH2 domains to microtubules, or at least a specific contribution of FH2 to microtubule interaction, has been reported for several metazoan formins ,43,44,45In addition to microtubule co-localization, we observed partial overlap of AtFH14-labeled structures with the endoplasmic reticulum, as well as peripheral association of AtFH13-labelled particles with the ER network. Some derivatives of the Arabidopsis Class I formins, AtFH4  and AtFHN. benthamiana epidermis differed noticeably, we nevertheless observed co-localization of a fraction of the signal, and documented that the FH2 domains of these two Class II formins can form heterodimers in the yeast two-hybrid assay. Crystallographic studies revealed that the functional form of formin’s FH2 domain is an antiparallel dimer capable of actin nucleation or barbed end capping . In the Heterodimerization of paralogous formins might theoretically provide a possible mechanism of formin activity regulation, as well as means of increasing functional diversity of fomins ; howeverArabidopsis lyrata or to Brassica sp. Only full-length sequences that retrieved the original query upon reverse BLAST search as the best match were considered. Missing exons in the predicted sequence of the AtFH13 ortholog from A. lyrata were added manually based on an experimentally sequenced partial cDNA (GenBank EFH40810). Multiple sequence alignment was constructed, manually cleaned of unreliably aligned segments and used to compute a maximum likelihood phylogenetic tree as described previously [For phylogenetic analysis, closest AtFH13 and AtFH14 relatives were identified by BLAST in the RefSeq section of GenBank after taxonomic restriction of the database to eviously  except tin planta were prepared as follows. Vectors were generated using conventional restriction cloning or the Gateway (GW) cloning system . For construction of entry clones, desired inserts were amplified using a PCR reaction with specific primer sets and templates summarized in TM1A (InvitrogenTM) for further construction of expression vectors. Amplified inserts were digested with appropriate restriction enzymes  vector was used. The PCR products were amplified by specific primers (TM). To obtain expression vectors, the resulting entry clones and binary destination vector pUBC-YFP-DEST/-RFP-DEST/-GFP-DES [TM). The combinations of entry clones and destination vectors are summarized in Constructs for transient expression  primers  and tran-GFP-DES , which aAgrobacterium tumefaciens strain GV3101 or C58C1. Plasmids carrying fluorescent protein markers, namely LifeAct-RFP [Nicotiana benthamiana  plants grown on peat pellets  under the 8 h dark/16 h light photoperiod at 22 °C were used. Overnight Agrobacterium cultures were diluted in the infiltration medium . A mixture containing equal amounts of bacterial suspensions carrying each construct of interest, corresponding to final optical density (OD600) 0.07 each, together with an amount of a suspension of agrobacteria expressing the viral silencing suppressor p19 [600 0.05, was infiltrated into the abaxial side of N. benthamiana leaves using a syringe. The fluorescent proteins were observed 48 h post-infiltration using confocal microscopy.For Agrobacterium-mediated transient transformation, expression vectors were transferred by electroporation into eAct-RFP , KMD-RFPeAct-RFP , ER-rk [eAct-RFP , were usssor p19  correspoFor the oryzalin treatment, oryzalin  was dissolved in dimethylsulfoxide (DMSO) as a 20 mM stock solution, stored at −20 °C, and later diluted to 10 μM final concentration with distilled water. Before observation, discs cut from infiltrated tobacco leaves were incubated in oryzalin solution (or in water for controls). Microscopic images were obtained using the Zeiss LSM880  confocal laser scanning microscope with a C-Apochromat 40×/1.2 W Korr FCS M27 objective. Fluorophores were excited with the 488 nm argon laser  and 561 nm laser (RFP), respectively, and detected by sensitive 32-chanell Gallium arsenide phosphide (GaAsP) spectral detector. All images were processed and analyzed using the Fiji software. All typical localization patterns that have been regularly observed in two to four independent biological replicates, each involving several leaves, are shown. Particle tracking was performed using the Fiji plugin TrackMate  v3.8.0 wConstructs for the yeast two-hybrid assay were prepared by restriction cloning from previously prepared AtFH13 and AtFH14 clones (see above) and introduced into vectors pGADT7 (containing the GAL4 activation domain) and pGBKT7 (containing the GAL4 DNA binding domain). Primers, templates, and destination vectors are summarized in


This study focused on identifying factors that motivate individuals to make provisions for their old age. We hypothesized that the motivation to make preparations would be associated with the importance attached to a specific domain of functioning along with the extent to which the individual experienced recent change within that domain. We examined how perceived changes in five different domains of functioning  during the last four years and the importance attached to each domain affected preparation for old age. Participants aged 35 to 85  were part of the Aging as Future Study. Across domains, perceptions of positive change and high importance were related to more engagement in preparations, as was being older. The strength of these effects as well as moderating influences varied across domains and cultures, reflecting potential differences in values and social/cultural supports.


The DEAH-box ATPase Prp2 is a key player during pre-mRNA splicing. Here, the first four crystal structures of this spliceosomal factor are reported in two different states, and the flexibility of its C-terminal domains and a previously undescribed ADP conformation are reported. act to the catalytically active B* spliceosome. Here, four crystal structures of Prp2 are reported: one of the nucleotide-free state and three different structures of the ADP-bound state. The overall conformation of the helicase core, formed by two RecA-like domains, does not differ significantly between the ADP-bound and the nucleotide-free states. However, intrinsic flexibility of Prp2 is observed, varying the position of the C-terminal domains with respect to the RecA domains. Additionally, in one of the structures a unique ADP conformation is found which has not been observed in any other DEAH-box, DEAD-box or NS3/NPH-II helicase.The DEAH-box ATPase Prp2 plays a key role in the activation of the spliceosome as it promotes the transition from the B In the subsequent step of the splicing cycle, the catalytically active B* complex is formed which facilitates the first transesterification reaction. After this, complex C is made by further remodelling and the second transesterification reaction takes place. Finally, the spliceosome is disassembled, leading to release of the spliced mRNA and the intron lariat.In eukaryotic cells, precursor messenger RNAs (pre-mRNAs) may contain noncoding intervening sequences (introns) that have to be removed by splicing prior to nuclear export and translation of the mRNAs. The process of pre-mRNA splicing is carried out by a highly dynamic multi-megadalton ribonucleoprotein (RNP) complex, the spliceosome, which catalyzes two sequential transesterification reactions  was identified by the NCBI BLAST search tool (GenBank EGS18341.1) and the gene encoding a truncated version of the protein comprising residues 270–921 was amplified by PCR from a total DNA preparation. The PCR product was cloned into the IBA Stargate pASG-IBA-25 vector according to the manufacturer’s protocol. The GST-fused recombinant protein was expressed in Escherichia coli Rosetta 2 (DE3) cells at 16°C. The cells were disrupted using a fluidizer (Microfluidics) in 50 mM HEPES–NaOH pH 7.5, 500 mM NaCl, 5%(v/v) glycerol, 10 mM EDTA, 1 mM DTT and the lysate was clarified by centrifugation for 30 min at 30 000g. The protein was purified at 20°C on a Glutathione Sepharose column  in 50 mM HEPES–NaOH pH 7.5, 500 mM NaCl, 5%(v/v) glycerol, 10 mM EDTA, 1 mM DTT. A wash step with binding buffer supplemented with 2 M LiCl was included to remove bound nucleic acids, followed by elution of the bound fusion protein with 30 mM reduced glutathione and cleavage of the GST tag with PreScission protease. Further purification was achieved using a Superdex 200 gel-filtration column, and residual GST was subsequently removed using a Glutathione Sepharose column. A check for contamination by nucleic acids was performed by measuring the absorbance of the protein solution at 260 and 280 nm. The protein was concentrated to 15 mg ml−1 using an Amicon Ultra centrifugal concentrator (Merck) in 10 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM DTT. A shortened construct comprising residues 286–921 was used for the crystallization of crystal form 3 (CF3), but all purification steps were performed as described above.Prp2 from 2.2.−1 (27.4 µM) and incubated with a tenfold molar excess of ADP or AMPPCP (274 µM). Crystals were grown at 293 K by sitting-drop vapour diffusion using drops consisting of 1 µl protein and 1 µl reservoir solution. Nucleotide-free crystals (NT-free) were obtained in 100 mM Tris–HCl pH 7.5, 200 mM Li2SO4, 15%(w/v) PEG 8000; crystal form 1 (CF1) in 100 mM HEPES–MOPS–NaOH pH 7.5, 60 mM MgCl2/CaCl2, 20%(w/v) ethylene glycol, 10%(w/v) PEG 8000; CF2 in 100 mM Tris–HCl pH 7.5, 200 mM NaCl, 15%(w/v) PEG 8000 and CF3 in 100 mM HEPES–NaOH pH 7, 250 mM NaCl, 25%(w/v) PEG 6000. Crystals were obtained after 2–5 d.Crystallization trials were performed with Prp2 at a concentration of 2 mg ml2.3.v/v) glycerol as well as 10%(v/v) PEG 400 for CF1 and CF3, and 10%(v/v) glycerol and 20%(v/v) PEG 400 for NT-free and CF2 prior to flash-cooling in liquid nitrogen. Oscillation images for NT-free and CF2 were collected on BL14.1 operated by the Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron-storage ring, Berlin-Adlershof, Germany  of 1.7 and a minimum CC1/2 of 60% for the highest resolution shell as cutting criteria  and simulated-annealing composite OMIT maps . For complete data-collection and refinement statistics, see Table 1The crystallographic phase problem was solved by molecular replacement, which was first performed for CF2 with 2.5.M and ADP/AMPPCP concentrations of 100 µM in the syringe. The reaction buffer consisted of 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 5% glycerol, 2 mM MgCl2. Each measurement consisted of an initial 6 µl injection and 19 injections with a volume of 14 µl injected at a speed of 1 µl s−1. The binding was monitored at 20°C and the interval between each injection was set to 250 s. Data integration was performed with NITPIC, integrated data were processed and analyzed with SEDPHAT, and GUSSI was used for the final representation of the data  using a Prp2 concentration in the cell of 10 µ3.3.1.Saccharomyces cerevisiae or Homo sapiens failed, the orthologue of Prp2 from the ascomycete C. thermophilum (ctPrp2) was identified and cloned, as proteins from this thermophilic eukaryotic organism may have a higher tendency to crystallize  as the 269-residue N-terminal domain is absent in the truncated protein used for crystallization. The helicase core comprises two RecA-like domains . RecA1 contains the conserved Walker motif A (P-loop) between residues 320 and 327. RecA2 contains a prominent antiparallel β-hairpin that protrudes out of the RecA2 domain. The winged-helix (WH) and helix-bundle (HB) domains are located C-terminally to RecA2; both are homologous to the corresponding domains in Prp43 and the Ski2-like DNA helicase Hel308  that range between 0.81 and 1.70 Å (the average r.m.s.d. based on six superpositions is 1.37 Å). This is significantly larger than the backbone r.m.s.d.s calculated separately for the RecA2 domain (residues 471–595 and 611–652), the helicase core comprising both RecA-like domains (residues 286–595 and 611–652), and the C-terminal domains , which range from 0.50 to 0.86 Å (average 0.65 Å), from 0.66 to 1.24 Å (average 0.97 Å) and from 0.44 to 1.03 Å (average 0.86 Å), respectively. Thus, keeping the RecA2 domains of the four compared Prp2 structures superimposed, the observed differences in the positions of their C-terminal domains  relative to the helicase core become more clear .Superpositions of the four Prp2 crystal structures presented here reveal distinguishable differences between their conformations, resulting in overall root-mean-square deviations (r.m.s.d.s) calculated between backbone Car Fig. 3a.b), are conserved among the four analyzed Prp2 structures regardless of the movement of the CTDs. The residues taking part in these conserved interactions are Arg657, Pro653 and Glu654, which form hydrogen bonds to Asn596 and Thr652 from the RecA2 domain; Pro719, Gly716, Glu654 and Asp679, which form polar contacts with Arg380, Arg401, Arg423 and Tyr461 from the RecA1 domain; and Arg423 and Lys435, which form salt bridges with Asp682 and Asp679 located in the RecA2 domain, respectively. This network of interactions between the winged-helix (WH) domain and the helicase core anchors the CTDs to the adjacent surface of the RecA1 domain. As a consequence, any movement of the RecA1 domain relative to the RecA2 domain triggers changes in the position of the CTDs, which form a single entity with the RecA1 domain. Surprisingly, comparison of the four Prp2 structures revealed that the observed changes in the position of the CTDs are not related to the presence or absence of ADP .The observed movement of the C-terminal domains (CTDs) with respect to the helicase core results in a different number of interdomain interactions (Supplementary Table S1). Interestingly, ten of these interactions, involving residues from the winged-helix domain  shows substantial differences in the conformation of the β-hairpin (residues 596–610), resulting in a significant increase in the calculated r.m.s.d.s, which range from 0.71 to 2.15 Å (average 1.61 Å) when compared with the r.m.s.d.s calculated between RecA2 domains without this flexible fragment . The observed conformational flexibility of the β-hairpin leads to substantial differences in the number of interdomain interactions formed between this structural element and mainly the winged-helix and OB-fold domains (Supplementary Table S1). The β-hairpins of NT-free and CF2 have similar conformations and protrude out of the molecular scaffold . The tips of these two β-hairpins were not resolved in the electron-density map and are probably disordered. In contrast, the β-hairpins of CF1 and CF3 are fully resolved and exhibit significantly different conformations and numbers of interdomain contacts (Supplementary Table S1). The largest number of interactions was observed for the deeply buried β-hairpin of CF3, which interacts mostly with the C-terminal domains.A superposition of the four compared Prp2 structures based on their RecA2 domains unveiled structural flexibility of the C-terminal domains and their coupled movement with the RecA1 domain. A closer look at the four superimposed RecA2 domains Fig. 4a shows ld Fig. 4a. The ta). Prp2 structures with the β-hairpin protruding out of the protein scaffold (NT-free and CF2) exhibit CTDs closer to the surface of the helicase core. Conversely, the structure with the buried β-hairpin (CF3) exhibits the most distant position of the CTDs relative to the RecA domains.A thorough analysis of the structural superposition of the four Prp2 structures reveals an apparent correlation between the conformation of the β-hairpin and the position of the C-terminal domains with respect to the helicase core Fig. 4a. Prp2 C. thermophilum Prp43. These structures exhibit significant conformational changes of the C-terminal domains which are related to their individual functional states .A comparable correlation can be observed in the structures of te Fig. 4b.3.4.2+ ion is coordinated by four water molecules, a β-phosphate O atom and the hydroxyl group of the Thr327 side chain.The two RecA-like domains form a cleft harbouring the bound ADP nucleotide. In the reported crystal structures of ctPrp2 complexed with ADP, the nucleoside diphosphates are bound by Gly323, Gly325, Lys326, Thr327 and Thr328. An Mgc). In CF1 and CF3 the adenine is sandwiched between Arg362 and Phe558 and the N6 atom forms a hydrogen bond to Ser358. The 3′-OH group of the ribose is involved in a polar interaction with Arg628. This conformation of the bound adenosine in the anti conformation in CF1/CF3 is very similar to that observed in Prp43–ADP crystal structures . In this new conformation (CF2) the adenine moiety forms two polar interactions with Asp582. The side chain of Arg362 is repositioned, now occupying the position of the adenine in CF1/CF3, forms a hydrogen bond to the 3′-OH group of the ribose. Superposition of the two ADP molecules (CF1 and CF2) based on their ribose moieties clearly depicts the actual differences in conformations . Although the sugar pucker remained unchanged (C2′-endo conformation), the torsion angles of the C4′—C5′ and C5′—O5′ bonds are changed in the CF2 conformation relative to those in CF1/CF3 by 167 and 62°, respectively. The observed conformational heterogeneity of ADP in the reported Prp2 crystal structures (CF2 versus CF1/CF3) reveals an intrinsic flexibility of the helicase core which is not related to large structural rearrangements or to crystal packing .In CF2, a novel conformation of the adenosine has been observed which is unique among all known DEin Fig. 5d. In thns Fig. 5e. Althod). A superposition of the RecA2 domains reveals that these loops in CF1 and CF3 adopt almost identical conformations and point towards ADP, whereas in CF2 this loop flips into a position more distant from the ADP. This alternate conformation of the loop enables the repositioning of the adenine as seen in CF2, as otherwise Arg628, which interacts with the ribose in CF1 and CF3, would hinder the novel conformation of ADP . Interestingly, this loop occupies a middle position in the nucleotide-free Prp2 structure when compared with the CF2 and CF1/CF3 Prp2–ADP structures.Additionally, the novel binding mode of ADP is accompanied by a change in the conformation of a loop at the C-terminal end of the conserved sequence motif VI Fig. 5d. A supDP Fig. 5c. Inter3.5.a). In contrast to CF1 and CF3, the motif VI loop is slightly flipped away from the ADP-binding pocket .The nucleotide-free Prp2 contains a sulfate ion that occupies the position of the β-phosphate in the ADP-complex structures Fig. 6. The O aet Fig. 5d.a). This finding is in agreement with the absence of AMPPCP in the nucleotide-free structure. The terminal O atom of the ADP in CF1–CF3 is involved in a hydrogen bond to Gly323 and this interaction could not take place with the methylene bridge of AMPPCP, which might explain the lack of binding of this ATP analogue. Binding studies via ITC were also performed for ADP as a control, and a dissociation constant (Kd) of 179 nM was obtained .The nucleotide-free structure was the result of crystallization trials of Prp2 with the nonhydrolysable ATP analogue AMPPCP. In order to explain the lack of bound AMPPCP, binding studies of AMPPCP and ADP were conducted using isothermal titration calorimetry (ITC). This experiment resulted in no significant heat signal with the buffer conditions used when AMPPCP was titrated. Although the lack of thermal signal might also arise from enthalpy compensation in the case of a deprotonation upon binding, this result strongly suggests that Prp2 does not bind AMPPCP . This is in agreement with previous biochemical data showing that Prp2 binds to the single-stranded intron ∼30 nt downstream of the branch-point site . The atomic models of both functional states share a very similar conformation and their overall structures strongly resemble the structure of the closely related DEAH-box helicase Prp43 Fig. 2. Howevera). Interestingly, the third ADP-bound structure (CF2) reveals a previously undescribed ADP conformation. In this structure the phosphates are bound in the same manner as in CF1/CF3, but the adenosine is flipped away from its position sandwiched between the RecA domains and points towards the RecA2 domain . This unique conformation is a consequence of changes in the torsion angles of the C4′—C5′ and C5′—O5′ bonds of 167 and 62°, respectively . Additionally, the adenine exhibits a syn conformation in CF2, in contrast to the more common anti conformation that is present in CF1 and CF3. The conformational changes of the ADP in CF2 seem to be accompanied by a flip in a neighbouring loop at the C-terminal end of motif VI and can only be sustained upon its conformational change, as otherwise the adenine would clash with Arg628 . Additionally, the more distal position of the loop slightly enlarges the entry to the nucleotide-binding pocket, which has also been observed in the nucleotide-free structure. The widening of the entry site to the binding cleft could play a role in the initial binding of ATP or the release of ADP, as it might make the binding pocket more easily accessible to the ATP molecule or promote the expulsion of ADP after completed hydrolysis. A further indication that the observed atypical ADP conformation might correspond to the ADP-prerelease state can be based on an analysis of the interactions between Prp2 and the adenosine moiety. Compared with the typical conformation, two hydrogen bonds (Ser358–N6 and Arg628–3′-OH), a π–π interaction and a cation–π interaction are exchanged for three polar interactions . The reduced number of interactions formed by the adenosine suggests a more loose association of the nucleotide with the helicase, which thereby also abandons its position in the interface between the two RecA domains. Thus, the Prp2 structure with the flipped ADP might correspond to a structural snapshot subsequent to the state observed in the other two ADP-bound states and prior to ADP release. The nucleotide-free structure could represent the state of Prp2 immediately after the release of ADP as well as shortly before the uptake of ATP. CF1 and CF3, on the other hand, display an ADP conformation that is already known from Prp43 structures, in which the adenine is sandwiched between an arginine from the RecA1 domain and a phenylalanine from the RecA2 domain  not only show a high overall structural similarity to the known post-catalytic state of the DEAH-box helicase Prp43, but also exhibit a comparable ADP conformation Fig. 2a. Interin Fig. 5d. This ly Fig. 5e. Additi are generated after ATP hydrolysis, and to date no exact order of events after this step has been assigned for DEAH-box helicases. For the DEAD-box helicase Dbp5 it was demonstrated that release of Pi is the rate-limiting step and is then followed by ADP release . However, on the opposite side of the RNA entrance the C-terminal domains need to be stably connected to the helicase core, and in Prp2 ten interdomain interactions were found that are present throughout all four structures . The WH domain is the only C-terminal domain that is involved in these conserved interactions and thus represents the most stable anchor point. Considering the facts that these common interactions are also conserved in Prp43 and that the observed flexibility of the C-terminal domains would support the current model of RNA binding, it seems likely that this mechanism might also apply to Prp2. Additionally, analysis of the Prp2 structures revealed a correlation between the position of the β-hairpin and the location of the C-terminal domains. The structures with a protruding β-hairpin exhibit a conformation of the C-terminal domains that is closer to the surface of the helicase core . This correlation suggests a passive role of the β-hairpin to be unlikely as it would otherwise be coupled to the movement of the C-terminal domains. Interestingly, a similar correlation can be found in the C. thermophilum Prp43 structures, although the conformational differences of the β-hairpins are smaller and the movement of the C-terminal domains is more pronounced . Based on structural comparisons with the Ski2-like DNA helicase Hel308 it has been proposed that this β-hairpin might play a role in duplex melting, but since no detailed unwinding mechanism for DEAH-box members has been described to date, the exact purpose of this structural feature remains unclear . Although it is involved in crystal contacts with two symmetry-related molecules, secondary-structure predictions from numerous servers agree on the presence of an α-helix between residues 270 and 296, as can be seen in CF1 , which is predicted to have a high α-helical content. The last 26 residues of this domain, which are present in CF1, also adopt an α-helical conformation. Interestingly, in human Prp2 (DHX16) the most N-terminal tip of the corresponding domain has been proposed to contain a PWI-like domain, which is also present in the spliceosomal helicases Brr2 (U5-200K) and has been predicted for Prp22 (DHX8) (Absmeier et al., 2015et al., 2012via substrate competition and conformational clamping (Absmeier et al., 2015et al., 2016et al., 2016The N-terminal extension of Prp2 has been shown not to be essential for its function, and the constructs used almost completely lacked this domain (Edwalds-Gilbert Despite the many parallels between Prp2 and Prp43 discussed in this structural study, the key question of why Prp2 is not able to act as an actual helicase cannot be answered using the presented structures. Prp2 and Prp43 are known to interact with so-called G-patch proteins and the regulation of their functions is tightly linked to this interaction. Detailed biochemical as well as structural characterization of this interaction might help to understand the peculiar manner of function of Prp2. However, most importantly, structural insights into Prp2 in a precatalytic state with bound RNA and an ATP analogue might reveal differences from the existing precatalytic structures of Prp43 which could shed light onto the as yet unknown mechanism of action of Prp2.5.et al. (2017et al. (2017The following references are cited in the Supporting Information for this article: Fica  al. 2017 and Liu  al. 2017. DEAH-box helicase Prp2, 6fa9PDB reference: complex with ADP, crystal form 1, 6facPDB reference: crystal form 2, 6faaPDB reference: crystal form 3, 6fa5PDB reference: 10.1107/S2059798318006356/ud5003sup1.pdfSupplementary Figures and Table.. DOI: