The same parameters significantly decreased as the flour particle size decreased from 25.81 µm to 10.15 µm (D50). Correlation analysis identified a significant negative correlation between flour particle size (D50) and damaged starch content and pasting temperature, while a significant positive correlation was found with the falling number values. The results of this work may have an important impact on the quality of processed foods.In the present paper, the properties of different ultrafine flour samples, including particle size distribution, damaged starch content, falling number, and pasting properties, were examined. The results indicated that the particle size decreased significantly after jet milling, as the rotation speed and grinding time increased, and the damaged starch content significantly increased as the size of the flour/starch decreased; this is in contrast to the significant decrease in falling number. Significant differences in pasting temperature were observed between straight‐grade flour (68.6°C) and five ultrafine flour samples (from 86.3 to 87.9°C). We also observed significant increases in peak viscosity, trough, breakdown viscosity, final viscosity, and setback as the flour particle size decreased from 43.07 µm to 25.81 µm (D The effects of ultrafine grinding on wheat flour/starch by jet milling was studied. Particle size and its distribution determine the properties of ultrafine wheat flour. Damage to starch granules was the most dominant factor influencing the final viscosity of flour/starch. Importantly, the methods aimed at reducing the production particle size will significantly alter the composition of the flour. For example, size changes result in structural damages and changes to the granule profiles as a result of the frictional heat and mechanical energy during roller milling, which further affects the functional properties and the quality of flour‐containing food. In their work, Scanlon et al.  observedIn addition to the wheat roll milling technique, superfine grinding is frequently used to decrease grain flour particle size and to improve the physicochemical properties of the fine powder, which influences the quality of the flour‐containing food  with a large surface area, high water absorption, and high solubility, which often result in more palatable foods. The objective of the present study was to investigate the effects of ultrafine grinding by jet milling on wheat flour, including the flour/starch particle size distribution, granule profiles, and the flour pasting properties. The overall goal of the study was to provide an important scientific contribution for the improvement of wheat flour quality.22.1The Zhengmai 9,023 cultivar samples were obtained from the Zhengzhou Haijia Food Company. The kernel hardness was 72%, and the protein content of the wheat samples was 14.8% .2.2The wheat cultivar samples (water content: 11.7%) were cleaned, sieved, and handpicked to remove impurities, and then, the moisture was adjusted to 16% over the course of 24 hr. The straight‐grade flour (SGF) was obtained by roller milling of the wheat samples using Bühler mill MLU‐202 (Bühler Group) according to the AACC method 26‐21A Experimental Milling—Bühler Method for Hard Wheat , with the flour extraction rate of 70.2%.2.31, UFS2, UFS3, UFS4, and UFS5. During the ultrafine grinding process, the UFS particle size was reduced by collisions among the flour particles or between the flour particles and the inner wall of the grinding bowl at a low temperature (0°C–5°C) and high pressure (0.75–0.80 MPa). Overall, we obtained five different UFS particle size distributions by adjusting the rotation speed  and number of grinding rounds (from one to three). The sample information is listed in Table Five ultrafine flour samples (UFS) were obtained by jet milling the SGF on the QYF‐100 jet mill : UFS2.4Different flour samples were thoroughly mixed, and the particle sizes were determined using a dry powder particle size Winner 3,000 detector (Jinan Winner Instruments Corporation). The instrumentation was equipped with a laser beam to detect the individual particles.2.5Microstructure of the SGF and UFS was examined by AMRAY1000B SEM analysis . The samples were attached to aluminum specimen holders with double‐sided adhesive tape and coated with gold under a vacuum for observation. The microstructure of each sample was observed at 20 kV with a magnification of 800× and 3,000×.2.6Damaged starch content of the SGF and UFS was determined according to a previously published method  according to the manufacturer's instructions. All determinations were performed at least three times, and the mean values were recorded.2.8The pasting properties of SGF and UFS were evaluated using a Rapid Viscosity Analyzer  according to the AACC method 61‐02. The pasting temperature (PT), peak viscosity (PV), trough viscosity (TV), final viscosity (FV), and the derivative parameters, including breakdown (BD = PV‐TV) and setback (SB = FV‐TR), were recorded. All determinations were performed at least three times, and the mean values were recorded.2.9The experimental results in this paper are reported as the mean of the results of two parallel experiments, and the Pearson correlation analysis was carried out to determine the relationship between different variables.33.150. D50 is the median diameter or median particle size and is commonly used to represent the average particle size of powders  was between 10.15 and 25.81 µm for UFS. The data suggest that jet milling resulted in a significant reduction of the average flour particle size with increasing milling speed and time.The particle size of SGF and UFS is summarized in Table Moreover, the results revealed that jet milling effectively transformed SGF into an ultrafine flour with particle sizes approaching 10 µm. As expected, the specific surface area was dramatically increased when the powder particle size was reduced. In their work, Zhang et al.  reportedThe ultrafine grinding technique is an emerging technology that can produce a narrow and relatively homogenous particle size distribution with good surface properties, decent dispersibility in foods, and excellent absorption in the body . For SGF, damaged starch content was 47.1%. These results suggest that the amount of damaged starch was created in the flour by the jet‐milling process. Starch damage is an important and well‐recognized criterion of flour quality ; this could be explained by the increase in contact areas with water . In this paper, we analyzed the properties of ultrafine flour from Zhengmai 9,023 samples. The data presented in this manuscript are the first to describe the effect of flour/starch granule profiles and particle size distribution on the FN value and pasting properties in ultrafine wheat flour. Our data clearly indicate that the particle size of flour/starch and its distribution are the dominant factors determining the properties of ultrafine wheat flour, including damaged starch content, FN value, and pasting properties. We discovered that the jet‐milling process significantly decreased the flour/starch particle size, which increased the damaged starch content; this may be directly responsible for changing the hydration characteristics and thermal properties of wheat flour. Our data suggested that when the particle size DIn summary, this work serves as a foundation for further studies examining other properties of ultrafine wheat flour, including changes in wheat starch/protein composition and the improvement of the rheological properties of wheat flour starch/protein, dough, and overall food quality. Additional studies would be beneficial to fully explain the effects of ultrafine milling on the grinding properties of wheat grain and promote improvements in the quality of final wheat products.None.The authors are grateful to Ke Bian for critically reviewing this manuscript. The authors are pleased to acknowledge Yuling Yang, Jinyue Pang, Tingjing Zhang, and Mengmeng Li for improving the communication of manuscript.This article does not contain any studies with human or animal subjects.


Compared to the PBCzA-L, the PBCzA-H with a higher molecular weight showed faster hole mobility and larger conductivity, leading to elevations in scJ via rapid hole transport, ocV via rapid hole extraction, and FF via lowered series and elevated shunt resistances. Thus, it is believed that PBCzA-H is a useful candidate for replacing liquid electrolytes.The leakage and volatilization of liquid electrolytes limit the commercialization of dye-sensitized solar cells (DSCs). As solid-state (ss) hole-transporting materials, free from leakage and volatilization, biscarbazole-based polymers with different molecular weights  and PBCzA-L (2450 g/mol)) were applied in combination with additives to produce ssDSCs. An ssDSC with PBCzA-H showed a better short-circuit current ( Howev2.95F0.05 doped with SnF2 [2SnI6 with additives [2(TFSI)2] and [Cu(tmby)2(TFSI)] imide) [2(TFSI)2] and [Cu(I)(dmp)(TFSI)]  [2 and [Co(bpyPY4)](OTf)3 ethyl)-2,2’-bipyridine and OTf is the trifluoromethanesulfonate anion) [2:dye/ssHTM/metal electrode (Au or Ag).Recent studies reported the photovoltaic properties of conventional liquid-junction-based ssDSCs ,6,7,8,9.Many carbazole derivatives have been applied as HTMs on organic electronics because of their excellent hole-transporting capabilities and chemical stability ,11,12,13In this study, biscarbazole-based hole-transporting polymers with different molecular weights were synthesized using procedures presented in the literature ,15,16,17N-carbazole (≥95%), N-2-ethylhexyl bromide (97%), iron (III) chloride (97%), 4-bromophenol (99%), potassium carbonate (≥98%), potassium phosphate (≥98%), copper (II) iodide (98%), trans-1,2-cyclohexanediamine (99%), sodium hydride , sodium tert-butoxide , tri-tert-butylphosphine (P(t-Bu)3, 90%), tris(dibenaylidene-acetone)dipalladium(0) (Pd2(dba)3), and N-bromosuccinimide (99%). Other chemicals for synthesizing polymeric HTMs were reagent grade and used without further purification. The following chemicals were purchased from Sigma-Aldrich : 2 pastes for the photoelectrode (Ti-nanoxide T/SP) and scattering layer (Ti-nanoxide R/SP), N719 dye (Ruthenizer 535-bisTBA), and hot-melt adhesive  were all purchased from Solaronix . TiCl4, LiTFSI, TBP, and 1-methyl-3-propylimidazolium iodide (MPII) were purchased from Sigma-Aldrich . We selected platinum paste  as the Pt counter-electrode source. An indium tin oxide (ITO) with 10Ω/square was purchased from AMG Co., Ltd. . A poly/poly(styrenesulfonate)  was received from H.C. Starck GmbH. All the chemicals for DSC fabrication were used without further purification. Commercial FTO (sheet resistance ~7 Ω/square) glass (TCO22-7), TiOThe biscarbazole-based hole-transporting polymers were synthesized using methods presented in the literature ,15,16,172:N719 dye and counter electrodes with a layer structure of platinized FTO/glass were prepared using the same procedures presented in an earlier work by this study’s researchers [Working electrodes with a layer structure of glass/FTO/TiOearchers . The cou−/I3−) with an ssHTM. An additive solution was prepared separately by dissolving LiTFSI , TBP , and MPII  in 1 mL of chloroform [2 layer and the platinized FTO layer, thereby fabricating liquid-junction-based ssDSCs with a 25 mm2 active area.Synthesized PBCzA (50 mg) was dissolved in 1 mL of chloroform to replace the conventional liquid electrolyte (Iloroform . The polAdditive-free hole-only devices were fabricated with a diode configuration of ITO/PEDOT/PSS (30 nm)/PBCzA (100 nm)/Au (70 nm) to estimate the hole mobility of the synthesized PBCzA with molecular weight. The same procedures presented in earlier work  were uti2:N719 dye/HTM with additives/platinized FTO/glass. Both mesoporous and scattering TiO2 layers were formed on a counter electrode, and the TiO2 was sensitized with N719 dyes. This electrode (glass/platinized FTO/TiO2:N719 dye) was sealed with another counter electrode (platinized FTO/glass) using a 25 μm thick sealing material. The mixed polymer and additive solutions were injected into the sealed cell through a predrilled hole on the counter electrode, and the cell was dried in a vacuum oven for 30 min at 50 °C. The injection and drying process was repeated four times to produce additive-containing hole-only devices.We also fabricated additive-containing hole-only devices with the layer structure of glass/platinized FTO/TiO1H and 13C NMR spectra were obtained using a Varian Unity Plus 300 spectrometer , and chemical shifts were recorded in ppm. UV–visible absorption spectra were recorded on a UV-2100 spectrophotometer , and the photoluminescence (PL) spectra were measured on a RF-5301PC fluorometer . The highest occupied molecular orbital (HOMO) energy level was measured using photoelectron spectroscopy . Molecular weight and molecular weight distributions of polymers were obtained using Waters gel permeation chromatography  equipped with Styragel HR 4E, Styragel HR 3E, and Styragel HR 1E columns while using tetrahydrofuran (THF) as an eluent against polystyrene standards at 40 °C. The 2) with a silicon photodiode . The dye-adsorbed TiO2 films’ active areas were estimated using a digital microscope camera  with image analysis software. All measurements were carried out under ambient conditions at room temperature.The cross-sectional morphology of the completed ssDSCs was visualized via field-emission scanning electron microscopy . The photocurrent–voltage measurement and electrochemical impedance spectroscopic (EIS) analysis were performed using a CompactStat potentiostat . A PEC-L01 solar simulator system equipped with a 150 W xenon arc lamp  was used as light source. The light intensity was adjusted to 1 sun (100 mW/cmSynthesized PBCzA polymer was split into high-molecular-weight (PBCzA-H) and low-molecular-weight (PBCzA-L) fractions via a repetitive reprecipitation procedure. The weight-average molecular weight and molecular weight distribution of polymers were measured at 21,200 g/mol and 2.6 for PBCzA-H and 2450 g/mol and 1.3 for PBCzA-L, respectively . The UV–scJ), open-circuit voltage (ocV) and fill factor (FF)) resulted in a higher PCE of 3.15 ± 0.43% for the device with PBCzA-H and the additives. We fabricated ssDSCs with PBCzA-H  and PBCZA-L (2450 g/mol) as HTMs and measured their photovoltaic properties to investigate the impact of molecular weight on the device performance. J) and voltage (V) curves of the ssDSC-H and ssDSC-L, and the device performance is compared in scJ, ocV, and FF compared to ssDSC-L. We examined the origins of the enhancement in all the photovoltaic parameters, and the analysis results are described in detail in the following sections.Revealing the superior PCE’s origins is essential, necessitating a focus on ssDSCs that show the highest PCEs among the four cells. Here, we denote an ssDSC (3.79% of PCE) with PBCzA-H by ssDSC-H and an ssDSC (2.73% of PCE) with PBCzA-L by ssDSC-L, respectively. scJ value increased by 9.54 mA/cm2 compared to ssDSC-L’s 7.53 mA/cm2, corresponding to a contribution of approximately 72% to the PCE’s enhancement. The diffusion length (dL) is the average distance that the excess carriers can cover before they recombine and can be expressed in the following equation: μ, Bk, T, τ, and q are the carrier mobility, the Boltzmann constant, the absolute temperature, the carrier lifetime, and the electron charge, respectively [By utilizing the PBCzA-H as a HTM, the ssDSC-H device’s ectively ,21,22.dL), which is proportional to the square root of the carrier mobility (μ), is one of the largest factors affecting scJ values in ssDSCs. Carrier mobility is particularly critical in liquid-junction-based ssDSCs because the distance between the top of the mesoporous TiO2 layer and the Pt counter electrode is about 7.1 μm  in the SCLC model is given in Equation (2): rε is the dielectric constant  of the polymer, 0ε is the permittivity of free space, h0μ is the zero-field hole mobility, L is the film thickness, E0 is the characteristic field, and V is equal to appl − (Vr + Vbi)V .The additive-free hole-only devices were fabricated using the PBCzA-H and PBCzA-L before their dark current–voltage characteristics were measured to estimate the effects of molecular weight on the hole mobility. Space-charge-limited current (SCLC) measurements have been used to evaluate charge mobility under steady-state currents in organic layers ,25,26. UJ–V data of the hole-only devices with a configuration of ITO/PEDOT/PSS (30 nm)/PBCzA (100 nm)/Au( 70 nm). This configuration can obstruct the electron injection from the Au electrode due to the large mismatch between the PBCzA’s LUMO energy level (2.27 eV) and the Au cathode’s work function (5.10 eV). In the logarithm of JL3/V2 versus the square root of V/L  and interface resistances. We observed three distinct semicircles corresponding to the resistances for carrier transport at the Pt/HTM (R1) and TiO2/N719/HTM (R2) interfaces and within the HTM (R3) in the ssDSCs. The fitted resistances using a Z-view software are given in l/(A3·R), where l is the thickness (around 22 μm) of the HTM between the two FTO glasses, A is the photoactive area (0.25 cm2) of ssDSCs, and 3R is the fitted resistance determined from the EIS measurements [scJ in the ssDSC-H could be attributed to higher hole mobility and electrical conductivity in the PBCzA-H layer than those in the PBCzA-L layer.Electrical conductivity can estimate carrier hopping in the ssHTM because it is proportional to the product of mobility and carrier concentration. EIS measurements of ssDSCs were performed to calculate the electrical conductivity. ocV value (0.590 V) was also higher than that of the ssDSC-L (0.566 V), which accounts for a 13% contribution to the PCE’s improvement. In solar cells based on organic semiconductors, ocV is generally written as Equation (3): n is the ideality factor, J0 is the reverse saturation current density, and ΔDAE is the activation energy for charge separation at the donor–acceptor interface [nterface ,30,31.ocV depends on the charge separation at the donor and acceptor interface (ΔDAE). Shu et al. reported that high mobility HTMs can extract holes easily from the donor layer, increasing the ocV [ocV value in organic solar cells with a configuration of ITO/HTM/donor/acceptor/buffer layer/Al and showed that a HTM with the highest mobility produced the largest ocV (1.15 V), whereas the other HTMs with lower mobility values exhibited a smaller ocV value. They attribute the increase in the ocV to a rapid hole extraction from donor to HTM, leading to effective charge separation (ΔDAE) [ocV in organic solar cells increased linearly with decreasing temperature and then saturated at lower temperatures (around 150 K), which was attributed to the temperature-dependent mobility of organic semiconductors [ocV. Elumalai et al. also found that ocV decreased with increasing disorder, which was attributed to the disorder-induced carrier traps decreasing the carrier mobility [ocV value is closely related to the carrier mobility, which affects the charge separation.Equation (3) shows that  the Voc . They aln (ΔEDA) . Elumalanductors . Conjuganductors , which cmobility . Thus, tocV value in the ssDSC-H was due to an effective charge separation (ΔDAE), resulting from the higher hole mobility than in the ssDSC-L.As mentioned earlier, the hole mobility of the additive-free hole-only device with PBCzA-H was about 290% higher than that of the counterpart with PBCzA-L. Moreover, the dark currents of the additive-containing device with PBCzA-H were higher than those of the device with PBCzA-L. Accordingly, a higher FF value (67.30%) compared to that of the ssDSC-L device with PBCzA-L (64.05%), corresponding to the 15% increase in PCE. The FF, defined as the ratio of the solar cell’s maximum power to the product of ocV and scJ, is affected by the series (seR) and shunt (shR) resistances [FF value in solar cells can be achieved by a decrease in seR and/or an elevation in shR [seR and shR values could be obtained from the slope of the J–V curves at ocV and scJ, as shown in Equations (4) and (5), respectively [The ssDSC-H device with PBCzA-H showed a higher istances ,34,35. An in Rsh . The Rseectively .(4)Rse=seR value (11.0 Ωcm2) in the ssDSC-H with PBCzA-H was recorded compared to that of the ssDSC-L with PBCzA-L (13.3 Ωcm2). Moreover, the shR of the ssDSC-H increased by 1238.4 Ωcm2 compared to that of the ssDSC-L (1100.1 Ωcm2). The seR value is closely related to solar cells’ internal resistance and can be partially estimated via EIS measurements. As shown in 2/N719/HTM (R2) and within the HTM (R3) were recorded in the ssDSC-H due to PBCzA-H’s higher hole mobility, contributing to the decrease in the ssDSC-H’s seR. shR is influenced by the charge recombination and the leakage current, i.e., the recombination could decrease the shR value in the organic solar cells [FF [2 ranged from 0.1 to 4 cm2/Vs, which is much higher than the organic HTMs’ hole mobility [shR value in the ssDSC-H. Thus, it can be concluded that the increased FF value was attributed to the lowered seR and the elevated shR by the increase of hole mobility in PBCz-H. As presented in ar cells . It is aells [FF ,37,38. Omobility . In this2 of scJ, 0.559 ± 0.006 V of oc,V and 68.18 ± 2.67% of FF, which led to a PCE of 2.51 ± 0.17%. In contrast, in the device with the higher-molecular-weight PBCzA-H , the PCE was increased to 3.15 ± 0.43% . The enhanced performance was attributed to higher hole mobility and electrical conductivity of PBCzA-H than those of PBCzA-L. Notably, the ssDSC-H showed the highest PCE of 3.79% even though the thickness of the hole-transporting layer (composed of polymeric PBCzA-H and additives) was over 7 μm. This result indicates that a higher-molecular-weight polymeric HTM is a better choice to obtain superior photovoltaic performance in ssDSCs. Polymeric HTMs with both biscarbazole and aniline moieties in the repeating unit were synthesized, and the molecular weight effects on the photovoltaic performance of ssDSCs were investigated. When the lower-molecular-weight PBCzA-L (2450 g/mol) was applied as a hole-transporting layer together with additives, the ssDSCs showed a photovoltaic performance of 6.60 ± 0.63 mA/cm


Modularity is important for evolutionary innovation. The recombination of existing units to form larger complexes with new functionalities spares the need to create novel elements from scratch. In proteins, this principle can be observed at the level of protein domains, functional subunits which are regularly rearranged to acquire new functions.In this study we analyse the mechanisms leading to new domain arrangements in five major eukaryotic clades  at unprecedented depth and breadth. This allows, for the first time, to directly compare rates of rearrangements between different clades and identify both lineage specific and general patterns of evolution in the context of domain rearrangements. We analyse arrangement changes along phylogenetic trees by reconstructing ancestral domain content in combination with feasible single step events, such as fusion or fission. Using this approach we explain up to 70% of all rearrangements by tracing them back to their precursors. We find that rates in general and the ratio between these rates for a given clade in particular, are highly consistent across all clades. In agreement with previous studies, fusions are the most frequent event leading to new domain arrangements. A lineage specific pattern in fungi reveals exceptionally high loss rates compared to other clades, supporting recent studies highlighting the importance of loss for evolutionary innovation. Furthermore, our methodology allows us to link domain emergences at specific nodes in the phylogenetic tree to important functional developments, such as the origin of hair in mammals.Our results demonstrate that domain rearrangements are based on a canonical set of mutational events with rates which lie within a relatively narrow and consistent range. In addition, gained knowledge about these rates provides a basis for advanced domain-based methodologies for phylogenetics and homology analysis which complement current sequence-based methods. Functional adaptations of proteins have often been observed to be caused by point mutations changing amino acids at crucial positions. These mutations typically result in altered specificity or stability of a protein. Although this process is important for evolutionary adaptations, point mutations often result in only minor changes of a protein. For greater functional changes or innovation, more drastic modifications are necessary that do not rely on numerous mutations.Pfam [Superfamily [Molecular mechanisms like crossing over, alternative splicing and transposition through mobile elements can cause mutational events that rearrange larger DNA fragments and therefore also alter larger regions at the protein level. Examples of such mutational events, which rearrange gene content, are for example fusion and fission. All these events lead to rearrangements that can be easily tracked at the level of protein domains, since domains are well characterised in many databases  and 20 In this study we use expanded species sets (up to 72 species per phylogenetic clade) to detect common patterns of domain evolution and consider several thousand more arrangements per clade compared to the two previously mentioned studies. In total, domain arrangements in five different eukaryotic clades  are analysed. For the first time, the results can be directly compared between these clades, since exactly the same methodology was applied to all of them.Previously, methods were applied that had used either overlapping definitions for rearrangement events, or that analysed domain loss and emergence (e.g. ) separatGene Ontology (GO) [Furthermore, we infer functional implications of the new arrangements via ogy (GO)  term enrfusion, fission, terminal loss/emergence and single domain loss/emergence; see Methods). For this purpose we reconstructed the ancestral domain content and arrangements at all inner nodes of the phylogenetic trees of five eukaryotic clades . For all domain arrangements that differ from the parental node, we examined whether the change could be explained uniquely by one of the six events.To be able to draw reliable conclusions about universally valid mechanisms in protein evolution, it is necessary to ensure that a sufficient number of observable rearrangements can be explained by the six different rearrangement events defined in this manuscript . Only unique solutions were further analysed in detail to focus on changes which can be explained with certainty . Beside these ambiguous solutions, some new arrangements cannot be explained by the defined single step events. These so-called complex solutions (25%-50%), would require several successive single step events.However, there is a small percentage of new arrangements which can be explained by multiple different event types, i.e. One major goal of this study is to find, beside clade-specific differences, universally valid evolutionary mechanisms of protein innovation that are present in all clades. Therefore, we analyse whether common patterns in domain rearrangements can be observed by measuring the relative contributions of each rearrangement event and compare them between the different clades , the most frequent domain rearrangement event is domain fusion (32% and 42% of rearrangements respectively), followed by single domain loss (27% and 20%) and terminal domain loss (21% and 19%). Arrangement gain by fission is slightly less common (20% and 17%), but still more frequent than the very low rates of single domain emergence (0.6% and 1.7%) and terminal emergence (0.1% and 0.4%).The functional analysis of gained arrangements in insects  and terms related to signal transduction . The fission of one arrangement into two new arrangements is the second most frequent mechanism (12% and 16%) followed by slightly smaller numbers of terminal (11% and 13%) and single domain loss (10% and 10%).Some GO terms are enriched in gained arrangements at the root of both plant clades that might be related to plant development and evolution, i.e. ’recognition of pollen’ in both plant clades or ’plant-type cell wall organization’ in eudicots Fig. .Fig. 2NWe analysed the distribution of domain arrangement sizes in the five clades , while fungi show the lowest duplication count (1.15).The general rates of rearrangement events and their distribution in a given phylogenetic tree can provide an insight into the evolutionary history of a whole clade as well as general adaptational processes in certain lineages. However, by taking a more detailed look at the specific domains involved in the rearrangement events at specific time points, we can trace back some major steps in the evolutionary history of the studied species. Here, we show three examples of new or outstanding functions at specific nodes in the evolution of vertebrates, plants and insects which can be related to the emergence of new domains or domain arrangements.One remarkable pattern in the distribution of rearrangement events in the vertebrate phylogeny is the high rate (33%) of single domain emergences at the root of all mammals. This represents the highest percentage of single domain emergences at any node in the vertebrate tree. A closer investigation of the function of these emerged domains shows that ∼30% of the emerged domains (domains of unknown function excluded) are associated with hair. This finding is a strong signal for the origin of hair or fur, respectively, in the common ancestor of all mammals.One of the most important structural protein families of mammalian hair is the keratin-associated protein family (KRTAPs). Hair keratins are embedded in an inter-filamentous matrix consisting of KRTAPs located in the hair cortex. Two major types of KRTAPs can be distinguished: high-sulfur/ultra-high-sulfur and high-glycine/tyrosine KRTAPs . Three oTriticeae which includes the two wheat species Triticum urartu and Triticum aestivum as well as the grass species Aegilops tauschii. Five out of the 15 enriched GO terms in Triticeae can be related to resistance to fungal pathogens via three different mechanisms. Chitinases are enzymes, which are known to be involved in plants’ fungal resistance and have been extensively studied in wheat species [Triticeae can be linked to the increased resistance of this clade to fungal pathogens.The functional analysis of gained domain arrangements using GO terms revealed an interesting pattern for the node leading to  species , 29. The species . The GO  species . Another species . We relaApidae, i.e. in the last common ancestor of the honey bee Apis mellifera and the bumblebee Bombus terrestris. This node marks one of the transitions of solitary bees to eusocial bees [Bombus terrestris and Apis cerana. Insulin and insulin-like signalling (IIS) pathways have been shown to be differently expressed between castes in the honeybee and play a role in caste differentiation [We found an example of interesting GO terms enriched at a node in ial bees . The oveial bees  and are ntiation , 35. Addntiation . Functiontiation , 35 and ntiation , 38. Sevntiation , i.e. ’Fntiation .In comparison to previous studies we can verify some of the key findings like fusions being the most common event type accounting for new domain arrangements , 20, 41.This increasing total number of resolvable events, while representing constant proportions over time, suggests that with increasing quality of sequences, annotations and motifs in databases we are able to explain more of the evolutionary history, but at the same time add more unknown or complex cases. However, the ambiguous and complex solutions we find in this study can be resolved to some extent with further investigation and approaches specific for this problem. In some cases, the ambiguity of ambiguous solutions might be resolved by computing domain trees based on the primary sequences. This is, though, outside the scope of this study and the information gain would be minimal as only a very low percentage (∼5%) of all solutions are ambiguous ones.Complex solutions might be resolved with the use of a deeper and denser phylogeny. Such a phylogeny might provide additional inner nodes which are required to be able to track the arrangement changes using single steps. Another potential way to resolve the underlying molecular rearrangement events of complex gains could be to consider not only single step events, but also solutions with two or more steps. However, the latter approach would strongly increase the complexity of the calculations, while at the same time introducing uncertainty by introducing multiple additional ambiguous solution possibilities.The GO term enrichment analysis based on domain changes during evolution can give additionally useful insights into major functional adaptations of a clade. In insects for example all described enriched GO terms  are essential for communication between individuals, for example to find mating partners by sensing pheromones over long distances or to tell nest mates from potential enemies in social insects –44. For One evolutionary mechanism of specific interest is loss of function as a process of adaptation. In this study especially the different signals for losses in plants and fungi are worth a more detailed investigation. In plants the high rates of fusion and fission and low rates of losses can be related to plant specific genome properties. Transposable elements play a major role in plants by the frequent creation of retrocopies and thus contribute to a high number of observable gene duplications in plants –47. AddiPuccinia graminis [Ustilago maydis [Saccharomyces cerevisiae. However, for Saccharomyces species there is evidence for an ancient whole genome duplication event followed by massive gene loss (an estimated 85%) of the duplicated genes [A possible explanation for the high frequency of single domain loss in fungi could be the generally high fraction of single domain arrangements in their proteomes. Such a high fraction of single domain loss is however not observed in plants, although eudicots also have a high fraction of short domain arrangements, comparable to that of fungi , insects (72), fungi (36) monocots (19) and eudicots (14)  is chosen. For each clade members of the clade itself are not used as outgroups, for example Drosophila melanogaster is not used as an outgroup for the insects. Strigamia maritima is additionally added as outgroup for the insect clade to make sure insect specific rearrangements are studied and not general arthropod rearrangements. In a first step all but the longest isoform of each gene is removed from the data set to prevent a bias in event rate detection by their influence on the analysis. Proteomes are annotated with Pfam domain models [Pfam. We used default parameters so that the script applies the thresholds specified in the Pfam database for annotating and filtering of the domains. Consecutive domain repeats in arrangements are collapsed to one instance of the domain .The reconstruction of ancestral domain arrangements and calculation of rates of domain rearrangement events is carried out using the in-house developed program ’DomRates’  arrangements , fission of existing  domain arrangements, loss of one or more domains (i.e. there are no traces left as the underlying DNA sequence is for example no longer transcribed) and emergence of one domain. The latter two biological events of loss and emergence can be divided into two different conceptual ones each. We distinguish in our study terminal loss/emergence and single domain loss/emergence, which can be both explained by the underlying mechanisms for loss and emergence. Terminal events describe the loss or emergence of domains at the ends of arrangements, while single domain events describe the complete loss or the first emergence of a single domain as a discrete arrangement. Terminal loss allows for more than one domain to be lost in contrast to just one domain considered for terminal emergence, since terminal loss can easily be caused by an introduced stop codon, which affects dependent on the position all following domains in the protein and not just the next or last domain. With this conceptual differentiation we make it possible to combine the two different approaches of previous studies (loss and emergence of single domains vs. reshuffling of domain arrangements).Since previous research in the field of protein domains focused mainly either on emergence and loss of single domains or on the evolutionary history of whole arrangements, sometimes postulating concepts such as recombination or domain-shuffling, it is necessary to specify the rearrangement events considered in this study see Fig. b. In facIt is important to note that all mutational events described here are defined purely on a domain level. On a DNA level different molecular mechanisms and mutations can lead to the same mutational event described here (e.g. fusion of two arrangements by fusion of neighboring genes through stop codon loss or through transposition of a second gene through mobile elements). For this reason we just define events we can infer explicitly on a domain level, while other potential molecular mechanisms leading to additional (less common) mutational events are not considered. An example for this would be the insertion of a domain/arrangement in the middle of an existing domain arrangement, which can happen through crossing over or transposition through smaller mobile elements, but cannot be distinguished on a domain level between insertion in the middle of an arrangement or two subsequent fusion events of independent arrangements. The possibility of multi step events or multiple possible solutions makes the definition of different solution types necessary.One can differentiate between four different solution types  from the rate calculation. A jackknife test with 100 repetitions is carried out by randomly removing 3 species from every clade and rerunning DomRates on the altered phylogeny to ensure robustness of the found rates and to identify possible sampling biases within clades. Means and standard deviation for every event type frequency in the jackknife test are shown in Additional file topGO package [Pfam domains to GO terms [topGOs ’weight01’ algorithm. Significantly enriched  GO terms are visualized as tag clouds.A Gene Ontology (GO) term enrichment is carried out with  package  in R. ThGO terms . The enrAdditional file 1 Rules of inference for both parsimony approaches. The middle panel shows which two parental states  for a domain or arrangement lead to which inference in the child node according to Dollo parsimony (left) or Fitch parsimony (right). The last line shows to what state an unknown state at the root is resolved.Additional file 2 Solution types. There are four different solution types by which a new arrangement can be explained. Exact and non-ambiguous solutions involve each just one event type .Additional file 5 Number of rearrangement events across the insect phylogeny. Digit representation of the total number of rearrangement events at a specific node is indicated next to the pie chart. For details on ‘Outgroups’ see Methods. Significant GO terms in gained domain arrangements are shown in a tag cloud (box). GO terms that might point to insect specific evolution are: chitin metabolic process, sensory perception of taste.Additional file 6 Number of rearrangement events across the vertebrate phylogeny. Digit representation of the total number of rearrangement events at a specific node is indicated next to the pie chart. For details on ‘Outgroups’ see Methods. Significant GO terms in gained domain arrangements are shown in a tag cloud (box). GO terms related to vertebrate evolution are strongly associated with regulation and signal transduction.Additional file 7 Number of rearrangement events across the fungi phylogeny. Digit representation of the total number of rearrangement events at a specific node is indicated next to the pie chart. For details on ’Outgroups’ see Methods. Significant GO terms in gained domain arrangements are shown in a tag cloud (box).Additional file 8 Number of rearrangement events across the monocot phylogeny. Digit representation of the total number of rearrangement events at a specific node is indicated next to the pie chart. For details on ’Outgroups’ see Methods. Significant GO terms in gained domain arrangements are shown in a tag cloud (box). GO terms that might point to monocot specific evolution are: ’recognition of pollen’.Additional file 9 Domain arrangement sizes. The size represents the number of domains an arrangement consists of, while the fraction relates to all discriminative domain arrangements in total for the specific clade. The total number of different arrangements considered in the data sets was 22199 (vertebrates), 22346 (insects), 10030 (fungi), 15565 (monocots) and 12097 (eudicots).Additional file 10 GO term enrichment analysis. Tag cloud for all events at the root of mammals in the vertebrate tree.Additional file 11 List of all species included in this study. Furthermore, for each species the related DOGMA completeness score of their proteome and version of the used genome assembly is shown.


Dioscorea opposita Thunb.) flour (from 0% to 25%) on the dough rheological characteristics, gluten structure, baking performance, and antioxidant properties of bread were investigated. The water absorption increased significantly (p < 0.05), while development time and stability decreased remarkably (p < 0.05) as the proportion of yam flour increased. SEM results indicated that the addition of yam flour destroyed the gluten network structure in the dough. Fourier Transform Infrared Spectroscopy (FTIR) spectra showed that addition of yam flour decreased the content of α-helix and β-sheet in gluten. With the increase in the proportion of yam flour, the specific volume and overall acceptability decreased (p < 0.05) whereas the total phenolics content (TPC), polysaccharides content, total flavonoids content (TFC), allantoin content, The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capability, fractal dimension, and hardness increased (p < 0.05). Overall, breads made of wheat flour replacement with no more than 15% Guihuai number 2 yam flour were of a high quality and had more antioxidant properties. These showed that Guihuai number 2 had broad application prospects in baked products.Impacts of wheat flour substituted with various levels of Chinese yam ( Dioscorea opposita Thunb.) has long been considered a food that improves health. In China, it is a traditional Chinese herbal medicine. Due to its high protein and starch content, yam is an important staple food in many subtropical and tropical regions and is the main source of carbohydrates and proteins [Chinese yam  which has been widely planted for food use in south China. Compared with other yam cultivars, Guihuai number 2 has the characteristics of high yield, small starch molecular weight, high amylose content, and low pasting temperature. Guihuai number 2 has not been fully utilized and well-studied compared to other yam cultivars. Evaluation of Guihuai number 2 for bread making is beneficial to broaden applications and thus promote the cultivation of yam. Therefore, it is necessary to evaluate the potential applicability of Guihuai number 2 as a partial substitution for wheat flour in bread making.Thus, the objectives of this study were to evaluate the effect of bread making substituted with Guihuai number 2 yam flour. The dough rheological properties and gluten protein structure were studied using farinograph, extensograph, rheometer, rapid visco analyzer (RVA), texture analyzer, scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). In addition, the bioactive components and antioxidant properties of bread were also investigated.Wheat flour was produced by Guangzhou Grain Enterprise Group . The composition analysis results as following (dry basis): 13.34% ± 0.04% moisture, 14.20% ± 0.10% protein, 69.87% ± 0.43% starch, 1.70% ± 0.05% fat, and 0.73% ± 0.02% ash content. Fresh yams (Guihuai number 2) were harvested from Baise city, Guangxi province. The yam tubers were washed, peeled, and sliced to 3mm thickness pieces. Yam slices were dried in a 101-2AB hot air dryer oven  at 60 °C [The method described by Nindjin, Amani, and Sindic  was usedFarinograph and extensibility were performed on farinograph and extensograph  respectively, with the method described by Li  and NindThe dough was cut into 50 × 40 × 15 mm cubes. Texture properties of dough were measured using texture profile analysis (TPA) on a TA.XT plus texture analyzer  with a P100 probe. The detailed condition was described in a previous study with slight modification . MeasureG′), viscous modulus (G″), and loss tangent (tan δ = G″/G′) were also determined [Dough rheological analysis was performed on a Kinexus Pro+ rheometer . Measurement conditions: parallel-plate geometry (20 mm diameter) with 1 mm gap, frequency range of 0.1–20 Hz and a temperature of 25 ℃. Furthermore, elastic modulus . The method was presented in previous reports ,16. MixeWater-washed gluten was prepared by using the procedure of Tuhumury et al. . The wetMicrographs of dough and gluten were visualized by a JSM-6380LV SEM . The freeze-dried dough and gluten samples were cut into 10 × 10 × 10 mm cubes. The samples were fixed, coated with sputtered gold and examined under SEM at an energy of 25 kV.−1, at a resolution of 4 cm−1, and 32 scans. Baseline correction was performed on the amide I band (1600–1700 cm−1) using peakfit 4.12 software . The Savitsk–Golay function was used for smoothing and deconvolution. The relative percentage of each secondary structure was calculated [FTIR measurements of gluten were performed on an IRAffinity-1 spectrometer . The absorbance spectra was recorded between 4000–400 cmlculated .Bread was cooled at room temperature for 2 hours and then cut into 12.5 mm thick slices. The crumb images were taken with a color camera. The crumb characteristics were analyzed by the method described by Gao et al.  and FarrCooled bread was accurately weighed, and volume was determined by the millet replacement method. The specific volume (mL/g) was calculated from the ratio of volume and mass of bread.Crumb texture was performed on a TA-XT2 texture analyzer . Strain was 70% , other cMoisture content was determined by heating sample at 105 °C for 4 hours in a 101-2AB hot air dryer oven . Fresh bread was used for test and the extract methods were described by Hsu et al. . Total pThe sensory panelists  were recruited based on their previous experience in the sensory evaluation of baked foods among postgraduate students and teachers of the Hunan Provincial Research Center of Engineering and Technology for Fermented Food, China. Sensory evaluation was determined according to a nine point hedonic rating scale method, the analytical procedure as described by Nindjin .p ≤ 0.05).All trials were carried out in triplicate and the data expressed by average ± standard deviation (SD) were analyzed by SPSS 22.0  and statistical significance was determined at 0.05 levels (p < 0.05) decrease of trough viscosity (from 1540 to 350 cP (1 cP = 10−3 Pa·s)), final viscosity (from 2737 to 745 cP), peak viscosity (from 2275 to 1285 cP), peak time (from 6.60 to 5.02 min) and setback (from 1198 to 395 cP), while the overall trend of breakdown showed remarkable (p < 0.05) increase. The decrease of viscosity values could be related to the composition, starch structure and mucilage of yam. Similar trends were reported that the viscosity values were significantly decreased by purple yam flour [p < 0.05). This is due to amylose–amylopectin ratio changes in these mixed flours, which had impact on the crumb structure and the specific volume of bread [The dough properties were evaluated using RVA, farinograph, extensograph, and texture analyzer, and the results were listed in am flour , and soyam flour  additionof bread .p < 0.05). According to Nindjin et al. [The farinographic analysis could reflect the rheological properties of the dough during its formation . After an et al. , yam stan et al. , potato n et al. . The incn et al.  reportedp < 0.05). According to Li et al. [The effect of yam addition on extensographic properties after 135 min was also outlined in i et al. . The addp > 0.05). Compared with W100Y0, hardness and chewiness of W95Y5 were largely reduced (p < 0.05). Hardness, adhesiveness and chewiness of dough showed a significant tendency for increasing first and then decreasing with the addition of yam flour from 5% to 25% (p < 0.05). Similar trends were also reported by Belghith Fendri et al. [As showed in i et al. . The higG′), viscous modulus (G″), and loss tangent (tan δ) of the dough. G′ was larger than G″ (tan δ < 1) at frequency range from 0.1 to 20 Hz. These phenomena indicated that elastic properties were predominated and the dough had solid, elastic-like features. The G′ and G″ of all dough samples increased as frequency increased. Similar trends were reported by Cao [The frequency sweep test was conducted to analyze the rheological properties of dough. d by Cao  and Baled by Cao .G′ and G″. Meanwhile, G′ and G″ increased as yam flour content increased. Dough W75Y25 showed higher G′ and G″ in comparison with dough with low level substitution of yam flour. These results indicated that substitution of yam flour significantly affected the dough structure and gluten network, and the influence became more pronounced as the amount of yam flour increased. Similar results were illustrated in studies of adding potato flour [G′ and G″ was different from that of Cao [G′ and G″. These results could be explained by the complex effects of protein, mucilage, and polysaccharides on dough viscoelasticity. During bread making, the rheological characteristics were dominated by the cross-linking of gluten network. Therefore, adding Yam flour to the dough, the gluten network had a higher crosslinking density. As shown in δ increased in the dough when the level of yam flour addition increased, which implied that the cross-linking degree in gluten network was reduced. Balestra [Compared to other samples, Dough W100Y0 had the lowest to flour . Furthert of Cao  who arguBalestra  reportedThe rheometer and texture data analyzer agreed with the farinograph results. These results revealed that the dough with yam flour substitution had a reduced capacity to keep its shape during proofing. Dough with yam flour substitution also decreased gas retention during the fermentation process, which affects bread porosity.−1 and 1682–1700 cm−1 were β-sheet characteristic peaks; 1637–1645 cm−1 was random coil characteristic peak; 1646–1664 cm−1 was the characteristic peak of α-helix; and 1664–1681 cm−1 was the characteristic peak of β-turn [−1. The content of secondary structure of gluten protein was listed in FTIR spectra of gluten were measured to clarify the effect of yam flour on gluten conformation . The secf β-turn . α-helixIt has been generally accepted that the most stable secondary structure was β-sheets. Similarly, an increasing in the α-helix would also lead to a more ordered structure . Accordi50 Value) of bread with different substitution ratio of yam flour were showed in Total phenolics contents (TPC), polysaccharides content, total flavonoids content (TFC), allantoin content and radical scavenging capability (ICAs shown in p > 0.05). When the addition of yam flour reached 10%, the fractal dimension was significantly higher than that of W100Y0. When the addition of yam flour was 25% (W75Y25), the fractal dimension of the bread was 1.80, which was an increase of 5.26% compared to W100Y0. The fractal dimension of the pore size distribution indicated the uniformity of the pore size distribution. The smaller the fractal dimension, the more uniform the pore size distribution. On the other hand, the larger the fractal dimension, the more uneven the pore size distribution and the greater the difference between the pore sizes. The increase of the fractal dimension indicates that the addition of yam flour changed the gas cell size formed inside the bread, and the difference in gas cell size increased. This may be due to the addition of yam flour, which affects the gas-holding properties of the dough. Crumb porosity affects the springiness and hardness of bread [The images of bread crumb were presented in of bread .p < 0.05). When the amount of yam flour was 25% (W75Y25), the bread hardness was 789.20 g, which was an increase of 67.10% compared with W100Y0. The increase in bread hardness was consistent with the change in dough hardness. Hardness had a greater effect on the overall score of bread, and had a negative correlation with bread quality. The smaller the hardness, the softer the bread. Springiness decreased remarkably with the increase in the amount of yam flour (p < 0.05). There was a positive correlation between the springiness and the quality of the bread. The larger the springiness, the better the quality. The decrease in bread springiness may be caused by the increased proportion of yam flour, the decreased gluten content of the mixed flour, the fact that the gluten network structure was destroyed, and the gas retention of dough was reduced. Good air holding resulted in decreased bread hardness and improved springiness. The tendency of bread cohesiveness and chewiness was consistent with hardness.It could be seen from p < 0.05) affected by yam flour addition, the highest specific volume was observed in W100Y0 and the lowest was in W75Y25. As Yam flour content increased, the specific volume decreased. This showed that gas holding capacity of bread had decreased during proofing and baking. This decrease would be due to destruction of gluten by adding yam flour, in agreement with the change of gluten network. A similar trend was observed by addition of other flours such as acha and bambara nut sourdough flour [The results of the effect of yam flour addition on specific volume were presented in gh flour , bran [3gh flour ,40, cassgh flour .p > 0.05). When the amount of yam flour was more than 5%, there was a remarkable difference between the overall acceptability of bread and bread W100Y0 (p < 0.05), and it decreased gradually when the substitution of yam flour increased (p < 0.05). When the amount of yam flour was not more than 15%, the antioxidant activity of the bread was high and the degree of acceptance was relatively high. The main reason was that the gluten content in the yam-wheat mixed flour decreased, the formation of gluten network structure was affected and resulted in a reduction in the sensory quality of the bread. Eke-Ejiofor J. et al. [As can be seen from . et al.  reported. et al. ,11. ThisG′ and G″ increased, while the peak time, viscosity value, setback, developing time, stability, and extensibility decreased. Moreover, as the proportion of yam flour was increased, the hardness, springiness and chewiness of dough decreased first and then increased. The microstructure of dough and gluten was destroyed, and the proportion of a relatively stable structure in protein secondary structure decreased by the addition of yam flour. In addition, the specific volume, hardness, and springiness of bread were all affected by changes to dough rheological and structural properties, following the addition of yam flour. The content of polyphenols, flavonoids, polysaccharides, allantoin, and the scavenging ability of DPPH free radicals in bread showed an increase which depended on the amount of yam flour. Compared with W100Y0, the addition of 5% yam flour didn’t exert significant negative impact on overall acceptability of bread. Acceptable bread was made by the substitution of no more than 15% yam flour for wheat flour in bread recipe. Adding Guihuai number 2 yam flour to bread recipe will improve the nutritional value and antioxidant capability of bread. The results of the present study demonstrated that Guihuai number 2 had broad application prospects in baked products.Replacement of wheat flour with yam flour remarkably affected the dough characteristics and bread qualities. With the increased percentage of yam flour, the breakdown, water absorption, degree of softening,


Protein domain-based approaches to analyzing sequence data are valuable tools for examining and exploring genomic architecture across genomes of different organisms. Here, we present a complete dataset of domains from the publicly available sequence data of 9,051 reference viral genomes. The data provided contain information such as sequence position and neighboring domains from 30,947 pHMM-identified domains from each reference viral genome. Domains were identified from viral whole-genome sequence using automated profile Hidden Markov Models (pHMM). This study also describes the framework for constructing “domain neighborhoods”, as well as the dataset representing it. These data can be used to examine shared and differing domain architectures across viral genomes, to elucidate potential functional properties of genes, and potentially to classify viruses. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12319631 E. coli, the best-studied bacteria, where one third of the proteome consists of proteins of unknown function. Here, we ask if (1) genomes can be decomposed into a series of functional building blocks that (2) do not rely on annotated genes and that (3) can be used to classify new species or genes, and if (4) protein domains can serve as these building blocks.Advancements in sequencing technology and the construction of large, publicly available genomic databases have widely expanded the potential for comparative genomics and discovery. But in viruses and bacteria, even protein-coding genomic regions are difficult to functionally characterize. Take domains provide just such building blocks and allow the decoding of some of this ambiguity across genomes. This approach will be based off of the identification of viral domains using profile Hidden Markov models (pHMM) with HMMER3 http://hmmer.org/, v3.2.11. Unlike sequence alignment, pHMMs are able to link two extremely divergent sequences that belong to the same type of protein domain. We referenced the profile databases PFAM2, vFAM3, and pVOG4. Although vFAM and pVOG have not been updated as recently as PFAM, they include many viral-associated domains not found in PFAM. The contents of these three profile-HMM databases form the “PFAM database” referred to throughout this manuscript. Here, we describe the construction of a reference-virus-complete, genome-wide, domain-based database. Domains are identified from the genome sequence, and domain-based “neighborhoods” are constructed. We describe this new dataset, comprising 9,051 viruses, and show some examples of novel queries to answer new biological questions that can be applied to any genome or set of genomes.Automatically defined protein 8. Dissecting the domains of novel proteins has led both to a better evolutionary understanding of the driving forces of the genes5, insights into taxonomic characterization and evolution10 and to the discovery of new enzymatic function11. Domain neighborhoods are also being used as tools for species classification6 and as an alternative to the standard taxonomic classification of 16s-rRNA sequence12. The success of domain-based classification in bacteria also has the potential to improve difficult viral classification, since there are no genes conserved across every virus.Domain-based approaches have been previously used in functional studies of mammalian genes, characterization and identification of pathogenic viruses, and phylogenetic analysis in bacteria13, bacterial pathogenesis15, and the study of temperature reactive domains16. Both GRAViTy (Genome Relationships Applied to Virus Taxonomy) and ClassiPhage 2.0 are tools for examining taxonomy using pHMM-based or genomic structural methods17. Another paper18, describes a new algorithm, MMSeqs. 2 for improving the throughput of the domain detection. Additionally, metagenomic data is difficult to analyze, and is sometimes simply converted to an approximation of species abundance. Instead, a domain-based approach allows for the preservation of the functional complexity within the metagenome, but with a simpler dictionary and a more complete analysis19, which we also enable with this work.A slew of recent papers has leveraged groups of protein domains to try to more broadly elucidate function. These include a secretion resourceftp.ncbi.nlm.nih.gov/genomes/Viruses/) a full list of accession numbers for the viral genomes used in this work has been included (Accession Number List20). The viral genomes include both eukaryotic and prokaryotic viruses spanning a wide range of viral families. While this reference file set will be used as an example, the domain-centric workflow is designed to be used with any set of sequence data, including genomic, RNA, protein, and metagenome. The overall pipeline is abstracted in Fig. https://gitlab.com/buchserlab/viraldomains).The data used to build these datasets were retrieved from publicly available sources. 9,051 viral genomes were downloaded from NCBI in the GenBank GBK and FAA format using the NCBI file transfer protocol (hmmsearch is sensitive to the length of sequence (target sequence) that is being searched. Our goal was to get a comprehensive look at all the protein domains, even allowing for some overlap (discussed later). Therefore, we used three different approaches to extract domains from each genome. 1) Whole genome search, 2) Gene search, 3) Window search. The whole genome search method simply feeds each entire contig (one of the 6 frames at a time) into hmmsearch. In the Gene-based method, we use the existing gene annotations to only feed the identified gene region to hmmsearch . In the Window method , each translated sequence is partitioned into overlapping 200 amino acid ‘windows’. No new sequence information is introduced during this process. Each 200 amino acid segment is then offset by 13 amino acids from the prior segment. As expected, feeding hmmsearch smaller sequences (as in the window method) increases its sensitivity to finding established domains compared with providing the algorithm with the entire genomics sequence. A comparison of the genome/window vs. the gene search method is done in the Technical Validation section.HMMER3’s 20). The vFAM and pVOG databases have been added in order to ensure that any viral domains not included in PFAM have been captured. This database also provides the profile framework for the HMM model2. Compiled FAA files are automatically examined to see if they have been previously run, then are copied onto a scratch location in a computing cluster. We then automatically generate new script commands, which run hmmsearch on a computing cluster .The domain profiles used in the pHMM model are from the PFAM, the protein family database provided by the European Bioinformatics Institute (downloaded 2/2018), vFAM, and pVOG databases, totaling 30,947 domains. A complete list of pHMMs is included , and FAAFile is the translated genome region that is currently being processed. For a set of genomes, 240 (6 frames x 40 pHMMs) are spawned and run in parallel on the cluster. Profile HMMs were bundled together in order to streamline execution and reduce computational burden. The resulting output files are monitored and if they are complete, they are moved to a different working directory . After processing has finished, the tracking logs are updated, but only for the correctly completed files, and the process is repeated, recovering any missing data. Next, the output files from hmmsearch (which contain the domain metadata) are compiled together to form a single large table . At this step, some filtering is performed. The per-domain independent E-values  are adjusted twice: first, they are scaled to account for the sequence search space size; second, they are scaled again linearly by the ratio of the viral genome size to the window size to adjust for the effects of the windowing. Domains with adjusted E-values > 1 are excluded. While the per-domain bit score and per-domain iEvalue (after accounting for search space size) provide nearly the same information, E-values were used because they are easier to scale, and E-values would most likely be more familiar to a potential researcher using this database. Finally, domains that are 100% overlapping are pruned down to a single copy.Where After compilation, the domains are automatically examined to remove spurious results . This is mostly from overlapping portions of domains and domains that are part of the same PFAM clan. The steps are, (1) Look at overlap on a per-frame basis (each frame separately), (2) Compare domain start/end and also the calculated start . 3a) If neighbor domains are in the same clan, only allow overlap of <33%. 3b) If domains are in different clans, keep both if each are significant; if not, then only allow overlap of <45% . (4) Consider nearest neighbor domains and ‘skip-1’ neighbors . In order to address overlapping domains from vFAM/pVOG overtop PFAM, additional logic was constructed. Only in the case that the log10 E-value of the vFAM or pVOG is five times higher than that of the overlapping PFAM domain is the vFAM/pVOG domain preserved.Next, we want to be able to ask questions of genome neighborhoods at the level of protein domains. Therefore, we want to map these domains onto the genome and reconstruct their ordering . There are several concerns to executing this correctly (listed in Usage Notes). The domains are ordered, and the user selects any number of “Domains of Interest”. These domains will act as the center of a genomic neighborhood, and the neighboring domains will list their coordinates in reference to this domain. This step produces the final dataset, and the tables are used to build the domain tracks, clustering, and other figures in the examples.b that is 4 domains away from Domaina in Virusi would get a value of 1/42 = 0.0625. If Domainb is missing in Virusi, that cell in the matrix gets a value of 0.The final dataset described above can be explored using a variety of clustering methods. In order to demonstrate this, we used a fuzzy clustering method, by keying the domains by their clan  and assigning weights to the domains based on their inverse square distance  as in Fig. https://figshare.com/. The first table is comprised of all accession numbers of viral genomes included (Accession Numbers20). Next, in (Trimmed Domain Compile File20) we provide the table of every domain within each of the reference viral genomes. Examination of domains in close proximity offers insight into conserved structure across genomes and commonly co-occurring domains. The method developed here allows domains found within a genome to be viewed within a “Domain Neighborhood.” The neighborhood comprises domains found in close genomic proximity to one another. This neighborhood is itself often a conserved unit, even when nucleotide sequence conservation is low, similar to genomic synteny, but without relying on primary sequence. The raw tables representing domain neighborhoods are available in (Domain Spacing File20). A lookup table containing column descriptors can be found in (Column Lookup Table20). A smaller version of the neighborhood file is available as a SQL database containing the necessary tables in (Domain Spacing SQL Database File20). Neighborhoods consist of a center domain of interest, which takes the zero position, and surrounding domains which have a negative or positive distance values based on whether they are upstream or downstream of the domain of interest, respectively. The structure of the data provided allows any domain to be used as the domain of interest, enabling the broadest spectrum of potential neighborhoods.The primary data is available in several tables, available on Domain Neighborhoods can be visualized using a domain “track” approach as shown in Fig. In order to ensure that the domains being identified by our approach are accurate and reproducible our pipeline was validated in two ways. The first validation approach was undertaken to ensure that domain annotation data was properly managed as it progressed through this pipeline. This is critical considering the large scale that HMMER3 is being run and the hundreds of thousands of output files that are produced during this process. In order to ensure that data integrity was maintained, we used the coordinates from the domain metadata of identified domains to extract sequence data from the FNA files. The sequences were reprocessed using the same pipeline to ensure that the same domains were identified. The extracted sequences yielded the same domains that were originally identified. Second, by doing gene/based domain extraction side-by-side with genome/contig based domain extraction, we were able to validate the identity of the domains Figs.  and 5.Fi20). In Fig. Contig-Based Domain-Finding Produces a Rich Set of Functional Domains. After running the pipeline on the set of reference viral genomes, we first sought to determine the completeness of the various methods. Both the translated genome method and the gene method yielded similar numbers of domains. Slightly more domains were found using the whole genome method Fig.  drawn frViral genomes are particularly interesting since they are known to perform double coding (overlapping reading frames). An examination of Human Papilloma Virus domains from this dataset showed Domain VFAM_11 and AAA_34 on the positive and negative strand as expected.21 of these two classifications is 0.53, showing that there is a high statistical correspondence between domain neighborhood-generated clusters and taxonomy. Newer approaches using these types of protein domains are generating exciting connections across biology22.The goal of this analysis is to redefine any sequence contig as a series of domains and be able to compare those sequences to determine whether they have shared or related domain neighborhoods. This can be used for a variety of purposes, and one of them is to help establish phylogenetic similarity. While this is not the focus of this manuscript, it provides another useful metric to validate the results. By comparing the clustering described above to virus families, we can create a contingency matrix, a scaled version of which is shown in Fig. 20), in addition to enabling domain comparison using widely present domains, allow for domain examination of family-specific and/or less common domains. This is made possible by using all available PFAM domain profiles as domains of interest. Figure domain of interest to examine a family-specific neighborhood. The flavivirus nonstructural protein 1 (NS1) is a glycoprotein that has a diverse set of functions during flavivirus infection impacting replication, immune evasion, and host vasculature disruption23. The wide range of roles attributed to this domain makes it a good candidate for further examination. Figure 24. This targeted approach to domain analysis allows the user of this dataset to gain insights into family specific domains to direct further research.The data from . Online-only Table While most of the viral genomes contained in the dataset have been assigned to known viral families, a small subset of the genomes analyzed were recorded as unclassified or unknown with regards to family membership, these will serve as an example of We leveraged “BI” software to visualize and organize the domain neighborhoods. We used Tibco Spotfire Analyst for this task, but Microsoft Power BI, Tableau, and other software can also effectively display these datasets. Additionally, Matlab or R (CRAN) can be used.Below we provide additional concerns on creating domain neighborhoods and domain-based approaches.Genome Completeness. We focused our efforts on complete ‘closed’ reference genomes so there would be no question of completeness. If extending these tools beyond viruses to bacteria, some species have additional chromosomes and plasmids which can house the genome neighborhoods. Our software also works on un-assembled genomes . In these cases, there can be some redundancy and there can also be missing information.Quality of Domain. We used hmmsearch to identify every possible query domain in the target sequence and reports the iE-value for the profile alignment. We store all these domains but set cutoffs when assembling genome neighborhoods. All domains with E-values less than ~10−7 are considered high quality, since a domain in a single genome would be considered high quality with an E-value of less than 0.01, and this threshold is divided by 9,051 to account for the size of the virome database.Overlapping Domains. There are two main types of overlapping domains in a genomic region. One type is inherent to the nature of similar domains given that domains in the same clan can often be detected in the same region. This is expected and easy to untangle by taking only the domain with the best E-value for an overlapping region. The second is the result of lower-quality domains being present, or very large domains which can have smaller domains nested inside them. We used the ‘de-overlap’ algorithm (in methods) to address this.Splicing, Ribosomal Slippage. Most viruses and bacteria have continuous coding regions, but introns do exist25. Although rare, it is also possible that a single domain is split across two frames due to ribosomal slippage26. Our program does not currently account for splicing or slippage, so these domains would be missed or would show up with an artificially low E-values (since they could be split up).


WRN is a key drug target for cancers with microsatellite instability. A crystal structure of the helicase domain of WRN identifies its unique features and provides a basis for structure-guided design of inhibitors. 2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core , comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional β-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn Werner syndrome helicase (WRN) is one of the five human members of the RecQ family of DNA helicases that unwind DNA in a 3′–5′ direction and play important roles in multiple pathways of DNA repair and maintenance of genome integrity . GermlinEscherichia coli RecA , together with three additional helicase associated domains in the C terminus: a zinc-binding subdomain (869–994), a winged helix (WH) domain  and a helicase and RNase D C-terminal (HRDC) domain . WRN is unique among the RecQ family of helicases in containing a 3′–5′ exonuclease domain in the N terminus (residues 38–236)  that share homology to domain 86–994, a w 38–236) , 11, the 38–236) , 13, witdomain 1,0–1,239.  38–236) . In homo 38–236) , 18. ConWRN was regarded as a “guardian of genome integrity” and a tumour suppressor. However, several recent studies have identified WRN as a potent and selective vulnerability of microsatellite instability-high (MSI-H) cancers , 20, 21.Previous studies , 21, 26 To address whether ATP hydrolysis is critical for WRN function in MSI-H cells, we introduced the Walker B motif mutation E669A, predicted to abolish ATP hydrolysis, into WRN. Subsequently, we compared the E669A WRN mutant with other mutant variants containing the Walker A motif mutation K577M predicted to prevent ATP binding, and the exonuclease-dead mutation E84A , 27, 28.Although our previous report showed that MSI-H cells fail to maintain the stability of their genome in the absence of WRN, it remained unknown whether ATP-binding and hydrolysis by the helicase domain of WRN was essential for genome integrity in MSI-H cells. To investigate this, we performed immunofluorescence analyses of the DNA damage marker γ-H2AX and scrutinized chromosome breaks in mitotic spreads. Expression of either the wild-type WRNr or the nuclease-dead WRNr E84A transgene prevented the increase in DNA damage marker . In striOur analyses of DNA damage markers and cell viability demonstrate that ATP-binding and ATP hydrolysis by WRN helicase, but not WRN exonuclease activity, are essential for maintaining cell viability and genomic integrity in MSI-H cells. The genome stability defects observed across the enzymatic WRN mutants strongly correlate with the observed loss of viability phenotype. This suggests that pervasive DNA damage elicited by loss of WRN protein or loss of ATP turnover by WRN helicase is responsible for the attenuated viability of MSI-H cells. Furthermore, our observations highlight the ATPase activity of WRN helicase as a therapeutic target to attack MSI-H cancers and the need for a crystal structure of the ATPase core of the WRN helicase domain. Our observations also provide pharmacodynamic markers of genome instability to track WRN ATPase inhibition in a cellular context.E. coli and a construct spanning residues 517–1,093 with a C-terminal hexahistidine tag and tobacco etch virus (TEV) protease–cleavage site. Crystallization was performed using sitting drop vapour diffusion and small shard-like crystals appeared between 1 and 2 mo. Crystals diffracted to 2.2-Å resolution, and the structure was solved by molecular replacement using a domain based search strategy with the structure of RecQ1 helicase (PDBid 2WWY) as a search model for the RecA domains (3AAF) as a search model for the WH domain  and is closely associated with the D2 domain. The WH domain extends away from the Zn2+ and D2 domains and features a modified version of the canonical WH fold with a longer wing 2.The overall structure of the WRN catalytic core consists of two RecA-like helicase lobes D1 and D2, each featuring a central six stranded parallel β-sheet flanked on each side by helices and loops . The Zn22+, aluminium chloride, and sodium fluoride, intended to produce the ATP analogue ADP-Aluminium fluoride, although examination of the electron density reveals only density for ADP , although the similarity to other human RecQ family members is only slightly lower   . One uni Å RMSD)  and S2C. Å RMSD)  in the WAnother notable difference between our structure of WRN and other RecQ family structures is the position adopted by the WH domain relative to the D2 domain. The positioning of this domain has been found to vary in other RecQ helicase structures, especially in the absence of DNA, whereas in the presence of DNA the complexes are more consistent, with the WH domain packed closely against the helical hairpin of the Zn domain , 35. In E. coli and Saccharomyces cerevisiae RecQ proteins indicated a role as an accessory DNA-binding domain, with the isolated HRDC domains having an electropositive surface and displaying binding affinity for single-stranded DNA (ssDNA) in the low to mid micro molar range , exhibiting higher solubility than WRN  under the experimental conditions. This effect could only be seen with the addition of high concentrations of WRN ATPase core (250 μM), indicating that the interaction of the unconnected HRDC and helicase domains of WRN is very weak under the experimental conditions. Sequential backbone assignment enabled mapping of the interaction surface onto the structure of the WRN HRDC domain. HRDC+Helicase/IHRDC < 0.4). The mapped interaction surface is in good accordance with the proposed model. The analysis suggests that the HRDC domain interacts with the helicase core and exhibits a similar spatial arrangement as described previously for human BLM. This weak interaction of the unconnected domains may be biologically significant in the context of the full-length protein, in which the WH domain and the HRDC domain are connected by a flexible linker of around 70 residues. This linker is significantly longer than in BLM (around 10 residues), and this additional flexibility may enable the WRN HRDC module to play other roles in addition to forming interactions with the helicase core.We have tested the possibility of interaction between the HRDC domain and the helicase core in solution by NMR using an 3AAF)   onto the to date . The firC. sakazakii RecQ in complex with an unwound G-quadruplex DNA, which found a guanine-specific pocket that accommodated a flipped out Guanine nucleobase with residues in this pocket being identified as essential for G4 unwinding  produced in insect cells lacking the WRN WH domain, which gave excellent peptide coverage of 91% (Table S1). As can be seen in HDX data summary and list of experimental parameters.Table S1 We have determined the crystal structure of WRN helicase core , a highly anticipated structure due to the recently discovered importance of WRN as selective dependency of and therapeutic target in MSI cancer cells. Our structure shows several unique features that may have implications in the WRN helicase mechanism. We show an unusual mode of nucleotide binding with extensive nucleotide interactions formed by residues in the D2 domain that have been confirmed in solution by HDX measurements. These interactions define the relative domain positioning of the D1 and D2 domains, which form a compact arrangement distinct from that seen in other RecQ structures and may represent a defined state in the WRN catalytic mechanism. Another possible feature of the WRN mechanism is an interaction between the WRN HRDC domain and helicase core. This interaction was demonstrated previously for BLM helicase, were the HRDC association ensures defined conformation of the helicase core via contacts to both D1 and D2. We show via NMR that a similar weak association between the HRDC and helicase core exists for WRN, and mapping of chemical shift perturbations onto the WRN HRDC structure indicates that the interface may be conserved. We have constructed a model for WRN DNA binding, in which the WH domain adopts an alternative position to that observed in the crystal structure. The model suggests possible roles for a WRN-specific insertion in the D2 domain and an unusual helical hairpin in defining the DNA protein interface.The human colon cancer cell line HCT 116 was grown in McCoy’s 5A medium with GlutaMAX  to which 10% FCS was added. Wild-type and mutant codon-optimized, siRNA resistant WRN transgenes containing a C-terminal 3xFLAG tag (designated WRNr) were synthesized and inserted into the lentiviral pLVX-IRES-puro plasmid vector (ClonTech) at GenScript. Lentivirus particles were generated using the Lenti-X Single Shot system (ClonTech) in 293T-Lenti-X cells. HCT 116 cell pools stably transduced with the WRNr transgene carrying lentivirus particles were selected with 2 μg/ml of Puromycin  added to the normal growth medium. These cell lines were generated using lentiviral transduction. Single cell clones were obtained by limiting dilution. All cell lines used in this study tested negatively for mycoplasma contamination and were further authenticated by short tandem repeat fingerprinting.Cells were lysed in extraction buffer  to which complete protease inhibitor mix (Roche) and phosphatase inhibitor cocktails  were added.The antibodies used in this study are as follows: WRN (8H3) mouse mAb , mouse anti-FLAG , rabbit anti–phospho-histone H2A.X (Ser139) , mouse anti-GAPDH , mouse Alexa Fluor 488  and secondary rabbit , mouse anti-IgG-HRP .For siRNA knock-down experiments, cells were transfected using Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen) supplemented with the following siRNA duplexes: WRN and PLK1 targeting ON-TARGETplus siRNA duplex ; ON-TARGETplus Non-targeting Control Pool . The final concentration of the siRNA was 20 nM in immunoblotting, immunofluorescence, and chromosome spread experiments. Cell viability experiments were carried out with 10 nM siRNA concentration in 96-well plates with a total volume of 100 μl per well and a starting number of 1,000 HCT 116 cells per well. Cellular viability was measured 7 d after transfection using CellTiter-Glo reagent (Promega). 100 μl of the 1:2 diluted CellTiter-Glo solution was directly added to the growth medium, mixed briefly, and incubated for 10 min before the measurement of the luminescence signal.https://www.indicalab.com/halo/) that identified DAPI-stained nuclei. Subsequently, the corresponding γ-H2AX mean intensities of the identified nuclei were determined.Cells were transfected as mentioned above and grown for 72 h. Subsequently the cells were fixed for 15 min using 4% paraformaldehyde, permeabilized for 10 min with 0.2% Triton X-100 in PBS and blocked for 45 min with 3% BSA in PBST (PBS containing 0.01% Triton X-100). Cells were incubated sequentially with primary antibodies that detect either FLAG or phospho-Histone H2A.X (Ser139) and secondary antibodies . Coverslips were mounted, and cells were counterstained on the glass slides using ProLong Gold with DAPI  (Molecular Probes). Images were collected using an Axio Plan2/AxioCam microscope and image processing was performed with MrC5/Axiovision software (Zeiss). Quantification of γ-H2AX foci was carried out using segmentation in the Halo software . To acquire chromosome spreads, cells in the fixative solution were dropped onto glass slides and air-dried. Slides were later stained with 5% Giemsa (Merck) for 4 min, washed briefly with tap water, and air-dried. The analysis was performed from two independent slides for each condition, and a blind quantification of the chromosome breaks was carried out. Images were acquired using an Axio Plan2/AxioCam microscope and image processing was performed with MrC5/Axiovision software (Zeiss).E. coli LOBSTR cells for overexpression  containing a protease inhibitor cocktail . The cell suspension was centrifuged at 27,000g for 40 min. The supernatant was loaded onto a Ni-NTA affinity column  equilibrated with lysis buffer and washed until baseline. Beads were treated with 20% elution buffer (lysis buffer plus 250 mM imidazole) to remove unspecific impurities and the protein was eluted with 100% elution buffer. The protein was cleaved by incubation with HIS-TEV protease at 4°C overnight. Afterwards, buffer was exchanged to lysis buffer, using a HiPrep Desalting column  and loaded again onto a Ni-NTA affinity column. Untagged Protein still possesses weak affinity to beads, thus a 10% elution buffer step was applied. The combined flow-through and 10% eluate fraction were concentrated by centrifugation, using an Amicon Ultra 30000 MWCO (Merck). The protein was finally loaded onto a HiLoad 16/600 Superdex 200 pg gel-filtration column  equilibrated with 50 mM Hepes, pH 7.5, 300 mM NaCl, 5 mM MgCl2, 1 mM TCEP, and 1 mM ATP. The major peak, corresponding to 48 kD in SDS–PAGE analysis, was concentrated to 15 mg/ml.WRN (residues 531–950) containing an N-terminal HIS-Zbasic-tag followed by a TEV cleavage site was cloned into a pFB-6HZB transfer vector. Recombinant baculovirus was obtained by transfecting Sf9 cells. Consecutively, Hi5 cells were infected with the virus and cultured in suspension using wave bags at 27°C for 50 h. Cells were harvested and disrupted by sonication in lysis buffer , the protein peak from the Heparin column was concentrated to 12 mg/ml using a 50,000 MWCO centrifugal concentrator diluted twofold in water  and co crystallized with 5 mM AlClms DIALS . The strms DIALS  with thems DIALS  and WRN ms DIALS  as startms DIALS  and the ms DIALS . A summaE. coli, strain BL21 (DE3). An overnight culture in Luria Broth supplemented with 100 μg/ml ampicillin at 37°C was prepared and added to M9 minimal medium supplemented with either 15NH4Cl (0.5 g/l), or 15NH4Cl (0.5 g/l) and [U-13C] glucose (2.5 g/l) the next day. At A600 of 0.95, the expression was induced by the addition of 0.25 mM IPTG and incubated at 20°C for 24 h (A600 of 3.1). Cell pellets obtained by centrifugation at 6,000g were stored at −20°C. Cells were solubilized in lysis buffer  and disrupted by sonication (Sonopuls from Bandelin) on ice. The sonicated lysate was clarified by centrifugation at 27,000g for 40 min. The supernatant was loaded onto a glutathione-Sepharose-4B affinity column  equilibrated with lysis buffer and washed until a stable baseline was obtained. The beads were mixed with TEV protease and incubated at 4°C overnight and washed with lysis buffer. The flow-through was concentrated by centrifugation using an Amicon Ultra 10000 MWCO. The concentrated solution was loaded onto a HiLoad 16/600 Superdex 75 pg gel-filtration column , equilibrated with 20 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM TCEP. The first major peak (containing TEV) was discarded and the second peak, corresponding to 11 kD in SDS–PAGE analysis, was concentrated to 11.4 mg/ml (15N-labelled HRDC) and 13.1 mg/ml (13C15N-labelled HRDC).The construct for expression of the WRN HRDC domain , as described previously , was obt1H-15N SoFast HMQC experiments were recorded in 3 mm NMR tubes (200 μl filling) at a protein concentration of 15N-labelled WRN HRDC of 30 μM ± unlabelled WRN helicase (531–950) 250 μM in sample buffer . Spectra were recorded on a Bruker Avance III 700 MHz spectrometer equipped with a cryogenically cooled 5 mm TCI probe at a 298 K and 128 scans, 128 f1 increments, and 2,000 data points in f2. Total acquisition time was 1 h.13C15N-labelled WRN HRDC of 1 mM in sample buffer  on a Bruker Avance III 600 MHz spectrometer equipped with a cryogenically cooled 5 mm TCI probe at a 298 K. The experiments performed included 1H-15N SoFast HMQC, 3D best-HNCA, 3D best-HN(CO)CA, 3D best-HNCO, 3D best-HN(CA)CO, 3D best-HNCACB, and 3D best-HN(CO)CACB  at a protein concentration of (CO)CACB . Spectra(CO)CACB .2O labelling, which was allowed to proceed from 10 s up to 4 h for each condition.Deuterium labelling: Deuterium labelling was initiated by diluting 3 μL of WRN  16-fold in deuterated buffer at room temperature. The labelling reactions were quenched by decreasing the temperature to 0°C and the pH to 2.5 by adding 48 μL of quench buffer. Quench buffer 1  was used for the AMP-PNP experiments and quench buffer 2  was used for binding experiments where ssDNA was present. Samples were taken at five time points . The sequence of the ssDNA used in the experiments was as follows: 5′-CCA GGT CGA TAG GTT CGA ATT GGT T-3′. Complexes with AMP-PNP and ssDNA were analysed in a similar way. WRN was incubated with AMP-PNP or ssDNA individually. Mixing ratios protein: AMP-PNP of 1:200 and protein:ssDNA 1:2.5 were used. The protein was allowed to equilibrate with the ligands for 2 min at room temperature before DUpon quenching, the samples were injected immediately into a Waters nanoACQUITY ultra performance liquid chromatography (UPLC) equipped with HDX technology. The samples were digested online using a Waters Enzymate BEH Pepsin Column  at 15°C. The cooling chamber of the UPLC system which housed all the chromatographic elements was held at 0.0°C ± 0.1°C for the entire time of the measurements. Peptides were trapped and desalted on a VanGuard Pre-Column trap  for 3 min at 100 μl/min. Peptides were then eluted from the trap using a 8–35% gradient of acetonitrile (with 0.1% formic acid) over 8 min at a flow rate of 40 μl/min and separated using an ACQUITY UPLC C18 HSS T3 1.8 μm, 1.0 × 50 mm column . The back pressure averaged ∼7,500 ψ at 0°C and 5% acetonitrile 95% water. The error of determining the deuterium levels was ± 0.15 Da in this experimental setup. To eliminate peptide carryover, a wash solution of  was injected over the pepsin column during each analytical run. Mass spectra were acquired using a Waters Synapt G2-Si HDMSE mass spectrometer. The mass spectrometer was calibrated with direct infusion of a solution of glu-fibrinopeptide  at 200 fmol/μl at a flow rate of 5 μl/min before data collection. A conventional electrospray source was used, and the instrument was scanned at 0.4 scans/second over the range 50–2,000 m/z with ion mobility engaged. The instrument configuration was the following: capillary voltage 3.2 kV, trap collision energy 4 V, sampling cone 40 V, source temperature 80°C and desolvation temperature 175°C. All comparison experiments were carried out under identical experimental conditions such that deuterium levels were not corrected for back-exchange and are therefore reported as relative.Peptides were identified using PLGS 3.0.1  using three replicates of undeuterated control samples. Raw MS data were imported into DynamX 3.0  and filtered as follows: minimum consecutive products: 2; minimum number of products per amino acid: 0.3. Peptides meeting these filtering criteria were further processed automatically by DynamX followed by manual inspection of all processed data. The relative amount of deuterium in each peptide was determined by subtracting the centroid mass of the undeuterated form of the peptide from the deuterated form at each time point and for each condition. These deuterium uptake values were used to generate uptake graphs and difference maps.6YHR. All HDX MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD018910.The crystal structure of WRN was deposited in the Protein Databank PDB ID