This work not only presents useful genomics methods for developing COFs and largely extended the COF structures, but also will stimulate the switch of materials development mode from trial-and-error to theoretical prediction-experimental validation.Materials genomics represents a research mode for materials development, for which reliable methods for efficient materials construction are essential. Here we present a methodology for high-throughput construction of covalent organic frameworks (COFs) based on materials genomics strategy, in which a gene partition method of genetic structural units (GSUs) with reactive sites and quasi-reactive assembly algorithms (QReaxAA) for structure generation were proposed by mimicking the natural growth processes of COFs, leading to a library of 130 GSUs and a database of ~470,000 materials containing structures with 10 unreported topologies as well as the existing COFs. As a proof-of-concept example, two generated 3D-COFs with  The discovery of new covalent organic framework (COF) topologies is often led by trial-and-error experiments. Here, the authors present a methodology for high throughput construction of COFs based on a materials genomics strategy and demonstrate the synthesis of the generated 2D and 3D-COFs. Such traditional approaches often result in lengthy time from initial research to practical application. In recognition of this, the United States in 2011 announced the Materials Genome Initiative (MGI) to accelerate advanced materials innovation, one of the main issues is to develop high-throughput computational tools for materials design to construct large materials database4. The subsequent programs with same ambition of Horizon 2020 and Chinese-version MGI were launched simultaneously in Europe and China. In the MGI, one of the significant challenges is to computationally construct vast space of possible materials for identifying optimal candidates. In this aspect, several pioneering studies have been reported in the literature6. An excellent example is the contribution of Snurr and coworkers7 to metal-organic frameworks (MOFs) that consist of organic linkers connected via metal ions9. They generated an impressive database of over 130,000 hypothetical materials using recursive, geometry-based algorithms to recombine a set of building blocks compiled from known MOFs. Alternative topology-based approaches were also developed in a top-down manner to construct massive hypothetical structures using underlying nets12. In addition to these hypothetical compounds, large archives of experimental MOFs were also built from the crystal data deposited in the Cambridge Structural Database (CSD)15.Advanced nanoporous materials are of great importance in various fields. Nowadays, materials discovery is primarily based on scientific intuition and expensive trial-and-error experimentation17. Depending on the structures of building units, COFs can be formed into those with either two- (2D) or three-dimensional (3D) structures20. In contrast to MOFs, the structures of COFs reported experimentally are very limited (~320), which is also the case in computational studies23. Apart from collection of experimental COFs24, a relatively large database was achieved by Martin et al.25, which contains 4147 hypothetical materials via framework interpenetration of 620 unique 3D-COFs assembled from their topology-based constructor. Recently, this method was further adopted by Smit and coworkers26 to greatly expand COF database. Since chemistry of COFs can be theoretically very rich, leading to these progresses still far from the need of computational screening from the viewpoint of the MGI. Furthermore, there is no large database available so far for 2D-COFs, although such materials have become a research hotspot in many areas27.Covalent organic frameworks (COFs) represent another emerging class of nanoporous crystalline materials distinguished from MOFs, which are assembled from organic reactants via covalent bondsTherefore, in complement with the library of nanoporous materials containing MOFs, our idea in this work is to develop relevant construction methods for COFs, particularly for 2D-COFs using a self-adaption algorithm to determine interlayer spacing. A database of ~470,000 COFs is built, from which four COFs are targeted synthesized as a proof-of-concept example, demonstrating the applicability of our materials construction methods. This work not only presents useful methods and a large COF database, but also gives an example for developing advanced materials based on materials genomics strategy.19, our partition method results in a total of 130 GSUs in the gene library of COFs (the library of GSUs is available from the authors as well as on the website: https://figshare.com/s/f8377d33bb9e9a73935a), possessing a variety of geometries, numbers of reactive sites and chemical compositions. Owing to the organic nature of COFs, these GSUs are further categorized into three types: center, linker and functional group)  was proposed. Firstly, to conveniently generate a variety of COFs, we adopted three different geometrical positioning methods to connect the reactive sites predefined on the GSUs, as shown in Supplementary Fig. 7, such post-functionalization operation in our QReaxAA on constructible parent frameworks can greatly increase the efficiency of high-throughput computational materials design. Finally, for large-scale construction of 2D-COFs with layered structures, special attention needs to be paid on how to appropriately arrange interlayer spacing. To address this issue, a self-adaption algorithm was proposed in this work. The general process of framework construction is given as follows.Besides the definition of materials genes, efficient computational tool for rapidly assembling COF structures on a large scale is another technical key in the viewpoints of materials genomics. Our genomic procedure shares much in spirit with the method of Wilmer et al.Parent structures of COFs are generated by stepwise connecting center- and linker-type of GSUs. The so-assembled frameworks are only allowed to contain one center-kind and at most two linker-kinds of GSUs. Once a success is achieved, chemical modification will be performed by attempting all kinds of GSUs categorized as functional groups. For 3D-COFs, both our approach and the approach by Wilmer et al. are straightforward, as shown in Fig. 26, which contains 61,199 3D- and 8,641 2D-COFs. It should be pointed out that our database at the moment only contains the structures featuring no mutually interdigitated frameworks.To check the validity of our genomic approach, we compared the experimentally reported COF structures with our generated ones and their energetically-relaxed counterparts by molecular mechanics (MM) optimization. For this purpose, six 2D-COFs and four 3D-COFs were taken as examples and the comparison metrics are their structural features which include cell parameters, surface area and void fraction, etc. Supplementary Tables mcm one is achieved by applying an existing  linkage to build structures, and the tth topology with two types of pores is realized via  linkage  and  linkages respectively leads to the sod and ffc topologies. In addition, the self-periodic extensions of the  and  linkages respectively generate structures with cds and cda topologies that possess continuous chains of center-type GSUs.The topologies identified for both 2D- and 3D-COFs are summarized as follows. For 2D-COFs, the age Fig. . For 3D-ig. Fig. : applyinhttps://figshare.com/s/c7e3b7610a71b9d64210.Some representatives of the generated structures with above topologies and the Bio-COFs generated using biologically-compatible GSUs are shown in Supplementary Figs. ffc topology which use the GSUs with tetragonal geometry as the core building units; such tetragonal GSUs exhibit a planar-like pattern with four connectivities, which could also be used to construct 2D-COFs28, in sharp contrast to those adopted for existing 3D-COFs. In addition, two 2D-COFs generated with existing hcb topology were also synthesized to enrich the chemistry of COFs. We noticed that among known linkages used for COFs, the most widely used one is imine bond due to its advantages such as high stability and more accessibility. More than two thirds of COFs reported in literature are fabricated through imine linkages and thus imine is more representative than other linkage models. Accordingly, imine was chosen as the linkage for the targeted materials synthesis.As a proof-of-concept validation to our materials genomics methods, targeted experiments were performed to synthesize some generated COFs. Compared to the dominant activity on the synthesis of 2D-COFs, expansion of 3D-COFs remains a significantly challenging task, especially for those with topologies unreported so far. All the existing 3D-COFs were solely reported for the nets based on the GSUs with tetrahedral or octahedral geometry, leading to the materials only with six distinct 3D topologies Table . In lighC4 + C3 linkage, the synthesis of the two 3D-COFs with ffc topology, a topology which has never been experimentally achieved before, was carried out by solvothermal reactions of 4,4′,4′′,4′′′-tetraaniline (ETTA) with 1,3,5-tri(4-formylphenyl)benzene (TFPB) or tris(4-formylphenyl)amine (TFPA), respectively, as shown in Fig. 3D-ETTA-TFPB displays two intense peaks at 2.20° and 3.84°, along with three peaks of relatively low intensities at 4.46°, 6.61° and 7.74°, which are assignable to the (010), (110), (020), (300) and (220) facets, respectively  and 4.25° (110), accompanied by several lower peaks at 4.91° (2-20), 6.55° (210), and 8.58° (220)  were performed  for 3D-ETTA-TFPB, and a = 41.8782 Å, b = 41.2827 Å, c = 17.8371 Å, α = β = 90°, γ = 120°  for 3D-ETTA-TFPA, nearly equivalent to those of the generated structures , 0, C4 + Chcb topology, which were generated using the GSUs both with C3-symmetric configuration. One material was synthesized by the reaction of 1,3,5-trimethyl-2,4,6-tris(4-formylphenyl)benzene (TTFB) and tris(4-aminophenyl)benzene (TAPB), while the other one was obtained by the condensation of TTFB and 1,3,5-tris(4-aminophenyl)amine (TAPA)  for 2D-TTFB-TAPB, and a = 24.4371 Å, b = 23.1107 Å, c = 5.6147 Å, α = β = 90°, γ = 120°  for 2D-TTFB-TAPA  Fig. . Similarata Fig. . RefinedThe above proof-of-concept experiments reveal the reliability of the materials genomics-based algorithms proposed in this work as well as its powerfulness. In contrast to an easy understanding of the topology of 2D-COFs by manually plotting the structures on the paper, it is hard to intuitionally figure out what architecture will be formed for 3D-COFs from different GSUs, especially in the finding of materials with unreported topologies. The above successful synthesis of 3D-COFs demonstrates the feasibility of our computational strategy in guiding experimental efforts to develop nanoporous materials.2 adsorption data in the range of P/P0 between 0.05 and 0.2  being 0.75 cm3 g−1 for the former and 0.77 cm3 g−1 for the latter. Their pore size distributions (PSDs) were estimated through nonlocal density functional theory (NLDFT), which display main distributions around 1.73 and 1.49 nm for 3D-ETTA-TFPB and 3D-ETTA-TFPA, respectively, indicating a good degree of crystallinity  and pore size to be 1526 and 2448 m2 g-1, 1.38 and 1.56 cm3 g−1, and 2.05 and 1.48 nm for 2D-TTFB-TAPB and 2D-TTFB-TAPA, respectively  was set equal to the largest contribution between center and linker GSUs plus that of functional group, as given bydcenter, dlinker and dfunctional group are the interlayer-spacing contributions of the center, the linker, and the functional-group GSUs that constitute the targeted structure, respectively.The covalently-bonded framework of 2D-COFs is restricted to 2D sheets, which are stacked together via van der Waals forces to form a laminar structure. Thus, there is no connection between the GSUs in adjacent layers. By examining the structures of synthesized 2D-COFs, we found that the interlayer spacing of most materials essentially is in the range of 3.0–4.0 Å. However, for some materials like IL-COF-134, COF-1035, COF-LZU136, CTF-137 and CTF-238) were selected and each of them was modified using the eight kinds of functional groups considered in this work. All the built structures were optimized using the Forcite module of Materials Studio. The final contribution of each functional group was averaged from its contributions to these five COFs, as listed in Supplementary Table The contributions of existing center and linker GSUs were determined from the interlayer-spacing information of the synthesized COFs. For other center and linker GSUs, their contributions were inferred from these derived GSUs with similar configurations. Since the functionalized COFs reported experimentally are very scarce, it is hard to establish the contributions of functional groups to interlayer spacing. With respect to this issue, we approximately set the contributions of them equal to the differences between the interlayer spacing of functionalized and parent COFs on the basis of the structures optimized using molecular mechanics. For this purpose, five 2D-COFs reported experimentally , pore-limiting diameter (PLD), accessible surface area (Sacc) and free volume (Vfree). In this work, accessible surface area of each material was calculated by a probe molecule with a size equal to the kinetic diameter of N2 (3.68 Å), while a probe size of 0.0 Å was applied to calculate the free volume which is the absolute amount of volume not occupied by the framework atoms. The void fraction (ϕ) was determined from the ratio of free volume to the total volume of the cell. The program used for the topological analysis is the open source tool TOPOS42.Structural features of the generated COFs in our database were analysed using the Zeo + + codeρcrys), pore-limiting-diameters (PLD), largest-cavity diameters (LCD), accessible surface areas (Sacc) and void fractions (ϕ). It can be found that these structural descriptors are in good agreement for each examined material. In addition, to validate the applicability of the interlayer-spacing contributions of functional groups used in our self-adaption algorithm, we arbitrarily selected 8 functionalized 2D-COFs that are not included in the training set and examined the variation of the cell parameters of each built structure after molecular mechanics optimization. As evidenced from the results shown in Supplementary Table c direction for each material changes only marginally, demonstrating that the contributions of the functional groups given in Supplementary Table To validate our genomic approach for the construction of COF structures, we compared some intrinsic structural features of 10 synthesized COFs (6 2D-COFs and 4 3D-COFs) with those of the built structures and the built structures after molecular mechanics optimization. Supplementary Table 26 Consequently, similar to the large databases computationally reported for MOFs and other materials, it cannot be guaranteed that all the constructed COF structures can be realized experimentally. However, apart from the offer of a theoretical guidance for the synthesis of COFs, the database also can provide a useful foundation for studying the structure-property relationships of COFs towards interested applications. Considering the issues described above, in the future we will manage to improve our algorithms to provide some useful insights into the synthetic conditions required to make COFs.Our method was mainly based on the monomers and the reaction types adopted in the synthesis of existing COFs, and thus all the structures were achieved through plausibly linking diverse monomers. In regard to examining whether certain COF structures that are likely to form, higher levels of theoretical approaches  were usually used in literature to explore their thermodynamic feasibility. However, not only is there a great difficulty in applying them to hundreds of thousands of COF structures in our database, but whether one material can ultimately be formed is also affected by kinetic factors relevant to solvents and synthesis conditions, which are difficult to consider in calculations.This COF was prepared by adding 4,4′,4′′,4′′′-tetraaniline (ETTA) , 1,3,5-tris(4-formylphenyl)benzene (TFPB) , chlorobenzene (3 mL) and acetic acid  (0.3 mL) into a glass ampoule. After being degassed through three freeze-pump-thaw cycles and then flame-sealed under vacuum, the ampoule was heated at 120 °C in an oven for three days. The resulting precipitate was collected through filtration, washed three times with anhydrous THF and 1, 4-dioxane, and immersed in anhydrous THF (10 mL) for 1 h during which the solvent was decanted and replenished with fresh THF three times. The product was filtrated and then dried under dynamic vacuum at 120 °C for 2 h to afford a yellow powder .3D-ETTA-TFPB from ETTA  and tris(4-formylphenyl)amine (TFPA)  in a mixture of chlorobenzene (2 mL) and acetic acid  (0.2 mL).This COF was prepared as an orange powder  using the same synthetic protocol of 3D-ETTA-TFPB from 1,3,5-trimethyl-2,4,6-tris(4-formylphenyl)benzene (TTFB)  and tris(4-aminophenyl)benzene (TAPB)  in a mixture of chlorobenzene (3 mL) and acetic acid  (0.3 mL).This COF was prepared as a light yellow powder  using the same synthetic protocol of 3D-ETTA-TFPB from TTFB  and 1,3,5-tris(4-aminophenyl)amine (TAPA)  in a mixture of 1,2-dichlorobenzene (2 mL) and acetic acid  (0.2 mL).This COF was prepared as a gold powder  using the same synthetic protocol of λ = 0.1542 nm). Each sample was spread on the square recess of XRD sample holder as a thin layer. Solution-state 1H- and 13C NMR spectra were collected on a Bruker Fourier 500 M or a 400 M spectrometer. Solid-state 13C cross-polarization/magic angle spinning (CP/MAS) spectra were collected on an Agilent DD2 600 Solid system equipped with a 3.2 mm HFXY MAS probe. The Hartmann-Hahn conditions of the CP experiment were obtained at a15 kHz MAS spinning speed with a contact time of 2.0 ms. Recycle delay times are 5 s. Fourier transform infrared spectroscopy (FT-IR) were obtained by using a Nicolet iS10 spectrometer.Powder X-ray diffraction measurements were carried out with an X’Pert PROX system using monochromated Cu/Kα  were activated by being immersed in anhydrous dioxane for 12 h. The solvent was decanted and the samples were dried under dynamic vacuum at 160 °C for 8 h. The resulting samples were then used for gas adsorption measurements from 0 to 1 atm at 77 K. The Brunauer-Emmett-Teller (BET) method was utilized to calculate the specific surface areas. By using the non-local density functional theory model, the pore size distributions were derived from the adsorption data.The code used to generate the COF structures are available from the corresponding authors upon reasonable request.Supplementary Information


The constitutive equation based on peak stress was established. Hot processing maps at different strains and recrystallization diagrams were also established and analyzed. The results show that dynamic recrystallization easily occur at high deformation temperatures and low strain rates. Safe and unstable zones are determined at the true strain of 0.6 and 0.7, and the hot deformation process parameters of partial dynamic recrystallization of the tested steel are also obtained.Hot deformation behavior of Fe-30Mn-0.11C steel was investigated. Hot compression tests were carried out at various temperatures ranging from 800 °C to 1200 °C and at different strain rates of 0.01 s Hot processing is an important metal forming process, and a combination of work hardening, dynamic recovery, and dynamic recrystallization processes ,2. The oThe Fe-Mn-C alloy system has been of interest for the development of materials capable of work-hardening to obtain very high strength levels ,7. The aIn the current work, the hot deformation behavior of the Fe-30Mn-0.11C steel including determination of hot deformation equations and hot processing maps, analyzing austenite recrystallization rule, and finding recrystallization region maps was studied. It provides a theoretical guidance for the industrial production of the studied alloy using hot processing.−1). During deformation, displacement and load were recorded for obtaining stress–strain curves. The specimens were water-quenched immediately after the deformation to preserve the high temperature microstructure.A high-Mn austenitic steel containing 0.11C, 30.5Mn (in wt %), which was coming from an experimental cast was selected for the present study where an ingot was produced from a vacuum induction furnace and subsequently forged in the temperature range of 800–1100 °C to form a bar with a diameter of 35 mm. Cylindrical samples with a diameter of 10 mm and height of 15 mm were cut from the hot-forged bar. A single pass hot compression test was carried out in a Gleeble 3800 thermo-mechanical simulator . The tantalum sheets coated with high-temperature lubricant were attached to both ends of a sample before a single-pass hot compression test, which ensured homogeneous deformation of the specimen and preventing the specimen from sticking to the indenter at high temperatures. A thermocouple was welded at the mid-height position of the cylindrical specimen for precise temperature control and measurement. The schematic representation of hot compression process is shown in Microstructures of the starting material and hot compressed specimens were characterized using a light microscope . For the investigation, cylindrical hot-compressed specimens were vertically sectioned through the center parallel to the compression direction, and a quarter on the section was observed. The sections were mechanically polished and etched using 5 vol % nital solution.A hot-forged high-Mn austenitic steel prior to hot compression  was usedThe true stress–strain curves at different deformation temperatures and various strain rates are displayed in T), and strain (ε) during hot deformation. The relationship between these parameters can be expressed using an appropriate constitutive equation. Flow stress analysis was carried out using a widely accepted Arrhenius type hyperbolic-sine relationship proposed by Sellars and Tegart [ασ < 0.8), high stress levels (ασ > 1.2), and all stress levels, respectively:Z) is the temperature-compensated strain rate factor [−1); A denotes the structural factor, α means the stress level parameter; n is stress index; β is a constant; Q is the apparent activation energy for hot deformation (kJ/mol); R represents the gas constant (8.314 J/mol·K); and σ could be peak stress –(6) are obtained:By taking the log function on both sides of the Equations (1) and (2) and partial differentiation at the constant temperature of ε versus lnσ and lnε versus σ are respectively plotted in n1 (n1 = 6.390185) and β (β = 0.064849) could be derived from the linear fitting of α (α = 0.010148) parameter could be obtained from Equation (6).lnT were respectively plotted in By taking the log function on both sides of Equation (3), the following Equations (7) and (8) are obtained and the following curves of n, Q, and A could be derived from linear fitting using T, which is shown in n = 5.645, Q = 393 kJ/mol, and Q was slightly higher than that of the high entropy alloy [Z). Therefore, the constitutive Equation (9) at σ = pσ could be obtained as:The values of py alloy  and simipy alloy . Figure θ(∂σ/∂ε) and flow stress σ, and the method is called the P–J method. In this study, the characteristic parameter values at different deformation conditions are obtained based on the P–J method, and the model reflecting its relationship with Z parameters is established as follows Equation (10). Where It is important to determine the hot deformation characteristic parameters and obtain its model . Poliak k and n are material Avrami constants, recovσ is the visual dynamic recover stress, and sσ is the saturation stress.The improved Avrami Equations (11) and (12) is used to describe dynamic recrystallization kinetics of materials :(11) XDRBy taking the log function on both sides of Equation (11), the relationship curve of k and n could be derived from linear fitting of n = 2.1586, k = 0.8369). Consequently, the dynamic recrystallization kinetic model could be obtained as:The value of DRXX at a temperature and strain rate. The obtained linear relationship (Equation (13)) makes it possible to predict Z parameters, and between deformation parameters, e.g., strain rate and deformation temperature are established and analyzed according to previous studies [Z parameter (Equation (14)) and a model expressing deformation temperature and strain rate (Equation (15)) are established:Since grain size largely determines the mechanical properties, such as hardness and toughness of the material, predicting product performance to establish the recrystallized grain size model is inevitable. The recrystallized grain size of materials at different deformation conditions is calculated using linear intercept method, and the relationship between grain size and  studies ,26. TherZ parameter being an intermediate variable.Z parameters and strain values as variables) is shown in Z parameter, which is a function of deformation temperature and strain rate, comprehensively indicates the influence of strain rate and deformation temperature on the mechanical behavior of hot deformation. Therefore, a recrystallization area map is an important tool for developing a reasonable hot processing process in industrial production.In order to reveal the relationship between deformation temperature, strain rate, strain and recrystallization percentage, and recrystallized grain size more clearly and intuitively, a dynamic recrystallization area map of the material based on the dynamic recrystallization kinetics model and the recrystallized grain size model was established. The dynamic recrystallization area map . The major results obtained were as follows:(1)A constitutive equation for the flow stress of the alloy at high temperatures was successfully obtained as a function of deformation temperatures and strain rates. The hyperbolic-sine of the flow stress showed a linear relationship with the Zener–Hollomon parameter.(2)cσ) with peak stress The estimated relationships between dynamic recrystallization critical stress ((3)Z parameter. The two models established were as follows:The accuracy of the recrystallization size model expressed by the deformation temperatures and strain rates was higher than that of the model represented by the (4)−1) with deformation temperature of 1000–1200 °C, which are the optimal hot deformation process parameters.A safe hot working zone locates in the low strain rate zones (about 0.01–0.6 s(5)−1, which are the optimal hot deformation process parameters.Fe-30Mn-0.11C steel possesses good hot workability and the finest grain size at a deformation temperature of 1000 °C and strain rate of 0.01 sThe hot deformation behavior of Fe-30Mn-0.11C steel was systematically studied using hot-compression tests at various temperatures (800–1200 °C) and strain rates (0.01–10 s


RpBphP1, in the bacterium Rhodopseudomonas palustris , has a C terminal PAS9 domain and a two‐helix output sensor (HOS) domain. Type 1 BphPs form head‐to‐head parallel dimers; however, the crystal structure of RpBphP1ΔHOS, which does not contain the HOS domain, revealed pseudo anti‐parallel dimers. HOS domains are homologs of Dhp dimerization domains in type 1 BphPs. We show, by applying the small angle X‐ray scattering (SAXS) technique on full‐length RpBphP1, that HOS domains fulfill a similar role in the formation of parallel dimers. On illumination with far‐red light, RpBphP1 forms a complex with gene repressor RpPpsR2 through light‐induced structural changes in its HOS domains. An RpBphP1:RpPpsR2 complex is formed in the molecular ratio of 2 : 1 such that one RpBphP1 dimer binds one RpPpsR2 monomer. Molecular dimers have been modeled with Pfr and Pr SAXS data, suggesting that, in the Pfr state, stable dimeric four α‐helix bundles are formed between HOS domains, rendering RpBphP1functionally inert. On illumination with light of 760 nm wavelength, four α‐helix bundles formed by HOS dimers are disrupted, rendering helices available for binding with RpPpsR2.Bacteriophytochrome proteins (BphPs) are molecular light switches that enable organisms to adapt to changing light conditions through the control of gene expression. Canonical type 1 BphPs have histidine kinase output domains, but type 3  RpBphP1 forms a complex with repressor RpPpsR2 when stimulated with far‐red light, causing a host of genes responsible for photosynthesis to be switched on. Photo‐isomerization of biliverdin IXα pigments causes long range structural changes from the far‐red (Pfr) to the red (Pr) absorbing state. These changes disrupt a four α‐helix bundle (magenta), making the HOS domain helix‐turn‐helix motif available for complex formation with RpPpsR2.The bacteriophytochrome  AppABlue light photo‐sensor proteinBphPBacteriophytochromeCBDchromophore‐binding domainc‐di‐GMPbis‐(3′‐5′)‐cyclic dimeric guanosine monophosphateDGCdiguanyl cyclaseDhphistidine kinase dimerization domainGAFGMP‐specific phosphodiesterases‐adenylyl cyclases‐FhlA domainHKhistidine kinase deomainHOStwo‐helix output sensor domainHTHhelix‐turn‐helix motifPASPer‐Arnt‐Sim domainPCDphotosensory core domainPfrfar‐red absorbing statePHYphotochrome specific domainPrred absorbing stateSAXSsmall angle X‐ray scatteringSECsize exclusion chromatography14=C15 bond. All BphPs have photosensory core domains (PCDs) at their N termini, comprising PAS‐GAF‐PHY domains, but can have variable C‐terminus output domains. PCD is the minimal structural unit necessary for stable and reversible photo‐conversion. Canonical type 1 BphPs have a histidine kinase output module that consists of a dimerization domain (Dhp) and a kinase domain RpBphP1 from Rps. palustris  XccBphP has also been determined ; however, this BphP possesses C terminus PAS9 domains only XccBphP and RpBphP1ΔHOS have been determined in their Pr and Pfr states, respectively, offering an opportunity to investigate light‐induced changes in type 3 BphPs. A major structural difference in XccBphP is that the long helix hI, which joins the PHY domain to PAS9, is bowed and the PAS9 domain is rotated outwards from the dimer twofold axis by ~ 30° XccBphP and RpBphP1ΔHOS is their different quaternary organizations ‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) by diguanylyl cyclase activity in the C terminus GGDEF domain Bacteriophytochrome proteins (BphPs) are photoreceptor proteins that transmit light‐dependent signals to downstream response regulators which affect a variety of light‐dependent physiological functions through the control of gene expression. The photo‐sensing molecule is a tetrapyrrole biliverdin IXα which is covalently attached to a cysteine residue near to the N‐terminus. Biliverdin IXα absorbs light and switches reversibly between the far‐red absorbing Pfr and red absorbing Pr state by isomerization of the D ring pyrrole about the CRpBphP1 is the transcription regulator RpPpsR2. This protein is found only in purple bacteria and is responsible for controlling the expression of photosynthetic genes. In purple bacteria, PpsR are loosely conserved, reflecting a high evolutionary divergence of properties Rhodobacter sphaeroides (Rba. sphaeroides) and Rhodobacter capsulatus (Rba. capsulatus), have revealed a common mechanism of binding to a pair of palindromic DNA sequences (TGTN12ACA) under oxidizing conditions Rba. capsulatus, PpsR is only redox sensing in Rba. sphaeroides, it is also antagonized by a blue light sensing protein AppA Rps. palustris and the closely related Bradyrhizobium sp., there are two such proteins, PpsR1 and PpsR2, but in most strains only PpsR1 has a Cys residue, whereas it is missing in PpsR2, indicating different redox behavior Rps. palustris strain CGA009, a laboratory strain that has been extensively used to study this bacterial species. CGA009 is not a typical strain because PpsR2 has the residue C428 and thus a similar redox behavior to PpsRs of Rba. capsulatus and Rba. sphaeroides. In CGA009, RpPpsR2 is under dual control, redox and far‐red light, through interaction with RpBphP1. However, all wild‐type strains studied so far have an R428 within RpPpsR2 and are not redox sensors. They are under the sole control of far‐red light, as will be discussed later The target protein of RpBphP1 and complex formation with RpPpsR2 will be presented here. Experimental findings indicate that full‐length RpBphP1 forms parallel dimers consistent with canonical BphPs and at odds with the structure of RpBphP1ΔHOS, which we conclude is a pathological dimer caused by HOS domain deletion. Based on the SAXS data, measured in the dark and under illumination with 760 nm light, models of RpBphP1‐Pfr and RpBphP1‐Pr are presented, and are subsequently employed to explain the role played by the HOS domains in the formation of native dimers and RpBphP1:RpPpsR2 complexes.Small angle X‐ray scattering (SAXS) experiments on full‐length RpBphP1 and 70 kDa RpBphP1ΔHOS fragment were prepared. The latter was purified by Ni affinity chromatography of protein, and is a natural degradation product when HOS domains are cleaved after approximately 3‐week period storage Illuminating full‐length RpBphP1 with light of wavelength 760 nm causes complete photo‐conversion to the red Pr state, which dark‐reverts to the Pfr state with a half‐life of ~ 50 min. The RpBphP1ΔHOS fragment exhibits incomplete photo‐conversion with a half‐life much shorter than 60 s  are ~ 220 and 160 kDa for Pfr and Pr states respectively  method. Given that RpBphP1 holo‐protein degrades to a 70 kDa fragment with the loss of HOS domains . A sample of 4 mg·ml−1 protein concentration placed in a 2 mm UV‐Vis cell path length was shown to undergo photo‐conversion to a 100% Pr state when exposed to λ = 760 nm light within 60 s  47.8/42.8 and Dmax 165 Å/140 Å for Pfr and Pr, respectively  0.65 and 0.82 respectively. The dimensions of Pfr and Pr dimers were 170 and 145 Å, respectively, tapering from 90 to 40 Å, and from 95 to 60 Å  which is also a bathy‐BphP with 32% sequence identity. To complete a full‐length structure, a HOS domain was built with the program modeller using four sequence‐aligned Dhp homologue X‐ray structures  of −1 to +1. An important feature of the optimized Pfr model is that HOS hJ′ helices must slide between hI helices which can form a short four α‐helix bundle, while helices hJ and hK form a longer four α‐helix bundle as observed in homolog Dhp dimerization domains of Histidine Kinases  toward the N terminus is required to obtain |ΔRg|/σ < 1. An unaccounted‐for feature in the Pr envelope is a ‘bulge’ situated between PHY and PAS9 domains which is capable of accommodating HTH motifs. A systematic search was performed of HTH orientations over the ‘bulge’ region using rearrangement about the hinge region while leaving hJ’ helices anchored to hI. The behavior of a number of models is illustrated in Fig. 2 values for each model as a function of ‘Angle’ and ‘Distance’. The angular displacement ‘Angle’ is defined by a line through the moving length of HTH relative to a fixed line joining the hinge region to helix hF in the PHY domain. ‘Distance’ is the corresponding spatial displacement of the turn T in HTH to hF. The top five models have χ2 =1.4−1.6 with HTH motifs approaching hF with ‘Angle’ in the 6°−12° range and ‘Distance’ in the 11 Å−16 Å range. The best Pr model analyzed in CRYSOL with the optimized hydration shell parameters yields χ2 = 0.94. A more extensive rigid body refinement of the HTH position, accessing ~ 200 000 models, provides a solution of χ2 = 0.7 which differs from manually refined models by a slightly larger HTH ‘Angle’ displacement of around 20°. Figure |ΔRg|/σ and χ2 values are reproduced to values that are less than 1.0. However, accurate positioning of HTH motifs in the ‘bulge’ region is not possible, as reasonable solutions can be obtained over an ‘Angle’ range of 6°–25°. The uncertainty in positioning HTH within the ‘bulge’ is to be expected because these motifs contribute only 14% to the whole structure and thus to the SAXS data.The Rps. palustris photosynthesizes anaerobically and, in strain CGA009, low oxygen conditions have been shown to weaken the binding of photosynthesis repressor proteins PpsR to specific DNA promoter regions. It has also been shown that far‐red light antagonizes binding of RpPpsR2 to DNA promoter regions on photo‐conversion of RpBphP1 to the active Pr state RpPpsR2 is observed to be predominantly a dimer in solution  and His6‐tagged RpPpsR2 (P) through a Ni2+ column shows that B does not bind, eluting at buffer imidazole concentration of 10 mM, while P elutes as two peaks, at imidazole concentrations P1 = 210 mm and P2 = 378 mm, corresponding to monomeric and dimeric protein respectively. A 4 : 1 ratio B : P mixture, exposed to 760 nm light, elutes at 10 mm imidazole as a slightly reduced B peak and a BP peak at 280 mm imidazole concentration  cannot form complexes with RpPpsR2, as confirmed by the absence of a 400 nm absorbing band at high imidazole concentrations  and ~ 50 kDa (RpPpsR2), respectively, as shown in Fig. RpBphP1:RpPpsR2 complex has a protein ratio of ~ 2 : 1  and the C‐terminus Dhp dimerization domain DrBphP from Deinicoccs radiodurans is a type 1 BphP exhibiting a Pr dark state. The photo‐conversion of DrBphP from Pr to Pfr states has been investigated by time resolved SAXS over the range 10 μs to 500 ms RpBphP1, where it is the Pr state that is destabilized, but this may only be a reflection of the fact that it is the dark states that are structurally stable in bathy BphPs.Bacteriophytochrome IsPadC has a diguanylyl cyclase (DGC) C‐terminus domain that modifies the second messenger molecule c‐di‐GMP. Structural asymmetry is observed in the X‐ray structure of IsPadCreg2 which has one monomer in the Pr state and the other in the Pfr state. Mutations that stabilize the mixed state were engineered within the 25‐residue coiled‐coil region (Reg2). In the Pfr protomer, Reg2 mutations have the effect of straightening the coiled‐coil, pushing its DGC domain closer to the two‐fold axis. This mutant is catalytically very active, suggesting that asymmetry may be important to its function. Comparing the structures of IsPadC and RpBphP1 shows that the Reg2 region maps directly onto the last 25 residues of helix hI in RpBphP1. Bowing of hI helices was observed in XccBphP1‐Pr suggesting that changes in conformation here is also important in type 3 BphPs. It has been proposed that asymmetry brings DGC monomers closer together, which renders the DGC dimer more active. The movement of Pfr DGC domain toward the two‐fold axis is similar to that observed for RpBphP1 where PAS9 domains are also closer to the two‐fold axis in Pfr than in Pr. It is interesting that IsPadC SAXS data of native protein show large changes in the Rg and Dmax, which are 49–56 and 160–170 Å for the dark Pr and the light Pfr states respectively. The SAXS data indicate that, in both IsPadC and RpBphP1, Pfr states form more elongated dimers than for Pr. Based on the UV‐Vis absorption spectra, which show incomplete photo‐conversion, a mixed Pr/Pfr state would be expected in the SAXS light‐illuminated data. However, a SAXS envelope has not been shown for this data. Such a calculation, if possible, would provide important information on the degree of asymmetry in the native protein.DrBphP has shorter Pfr dimers but Pfr dimers in both IsPadC and RpBphP1 are more elongated. This is despite IsPadC and DrBphP both being Pr dark stable, whereas IsPadC and RpBphP1 have Pr and Pfr dark stable states, respectively, yet exhibit similar SAXS Pfr properties. Structural differences in Pr and Pfr states probably also depend on the very different functional requirements of C terminus domains which are; for type 1 DrBphP a movement of Kinase domains toward the Dhp, in type 5 IsPadC it is the bringing together of DGC domains to make catalytic active dimers and in type 3 RpBphP1 a separation of HOS domains to facilitate the formation of complexes with RpPpsR2.Although comparison between different BphP types is useful, there are structural differences within the PCD that complicate matters. For example, the canonical dark‐stable Pr state, in type 1 BphPs, has bowed helices joining PAS‐GAF to PHY domains resulting in a large hole, whereas in other BphP types these helices are relatively straight, causing closer contacts within the dimer spine. It is interesting that the canonical ab‐initio reconstruction of envelopes. However, in a real‐world situation, light will be distributed across the full red to far‐red energy spectrum. There is, therefore, no reason to suppose that mixed states could not exist for RpBphP1. One possible model of the complex will be presented below, which could apply to both pure Pr dimers and mixed Pfr:Pr dimers.SAXS experiments were designed to obtain pure Pfr and Pr samples and to enable easier data analysis and RpBphP1. Although precise details on HOS contacts cannot be determined using the SAXS data, modeling nevertheless suggests that hJ′ helices must overlap with hI helices so that the long four α‐helix bundle can fall within the Pfr envelope. An interesting feature of our Pfr model is that helix hJ is brought in line with helix hI, creating a spatial helical topology that is similar to type 1 BphP, albeit with a break between these helices caused by an intervening PAS9 domain. The Pr model best fits the SAXS data when helices hJ and hK occupy the bulge region between PHY and PAS9 domains and this can be achieved through the hinge region which allows helices hJ and hK to articulate independently while leaving helices hJ′ in situ to form a short four helix bundle with hI helices. The Pfr model is consistent with the observation that the Pfr state is functionally inert, and we propose that this is because the long four α‐helix bundle confers structural stability to the HOS dimer while making HTH motifs unavailable for complex formation with RpPpsR2. Alternatively, our Pr model has free hJ and hK helices that can interact with RpPpsR2. The corollary of this is that an HOS deleted construct is incapable of forming complexes with RpPpsR2.HOS domains are essential for the assembly of head‐to‐head parallel dimers through the formation of a C‐terminus four α‐helix bundle that has the same dimer formation function as do Dhp domains in type 1 BphPs. This similarity may point to a common molecular ancestor, for example, type 3 BphP may have evolved from a type 1 BphP by insertion of a PAS9 domain and the loss of a kinase domain with additional point mutations that change Dhp domains to HOS domains. An important difference is that α‐helix hI is continuous with Dhp helices in type 1 BphPs XccBphP‐Pr, the movement of PHY domain toward the C terminus causes curvature of α‐helix hI and a rotation of the PAS9 domain away from the twofold axis 2, as shown in Fig. RpPpsR2.There are a number of studies characterizing photo‐induced structural changes in the PCD domain which can be used to propose a tentative mechanism for HOS domain activation in the Pr state. Light excitation of biliverdin IXα causes isomerization of the D pyrrole ring and rearrangement of amino acid residues in the biliverdin pocket RpBphP1:RpPpsR2 complex model that is consistent with experimental results, although the model presented here should be treated with caution until hard structural data become available. Nevertheless, it is worthwhile discussing the constraints that observed data can impose on complex models. These constraints are: (a) the formation of a complex with a protein ratio RpBphP1:RpPpsR2 of 2:1, (b) a central role played by HOS domains in the formation of complexes which also explains why RpBphP1ΔHOS cannot make complexes, and (c) complexes must be able to form with the Pr and not with the Pfr form of RpBphP1. A model was built based on these constraints and from the full‐length structures of RpBphP1 and RpPpsR2. A full‐length model of RpPpsR2 was based on a homolog partial structure RsPpsRΔHTH (PDBID 4HH2) from Rba. sphaeroides which represents ~ 83% of the full‐length molecule and has ~ 28% sequence identity with RpPpsR2. The missing part of the structure is the HTH domain at the C terminus belonging to the FIS family of DNA‐binding proteins which form four α‐helix bundles. A number of homologous HTH domains were found using the online toolkit HHpred and these were used to build RpPpsR2 in Modeller  and the other from an RpPpsR2 monomer (HTHP). Two forces are involved in the formation of four α‐helix bundles, electrostatic anti‐parallel helix dipoles and hydrophobic forces between inner α‐helix surfaces. These inner surfaces are usually lined with small hydrophobic residues, such as Leu, Ala, Ile, and Val. However, homo dimers must separate for these hydrophobic surfaces to be available for interaction and so that hetero dimers are formed. This is the case in the RpBphP1‐Pr model as HTH motifs are bent back whereby hydrophobic surfaces point outwards  which do not have a redox Cys in the HTH domain and are thus non‐redox sensing forms of PpsR. Consequently, in most strains, RpPpsR2 is normally only under the control of light by interaction with RpBphP1. It has been suggested that CGA009 is a laboratory strain which has acquired a frame‐shifted bphP1 and an R428C RpPpsR2 mutation over time due to laboratory selection of pigmented bacteria under certain laboratory conditions, which highlights the pitfalls of using laboratory strains RpBphP1 and an RpPpsR2 in reducing conditions is therefore a good model of complex formation in wild‐type strains. Although wild strains of RpPpsR2 are not redox sensors, in Rps. palustris, photosynthesis remains under redox control through other means. For example, the cytochrome c2 encoding gene cycA transfers electrons to the Reaction Centre from outside the cell and the redox controlled transcription regulator encoding gene aerR has promoter regions which are recognized by PpsR2. Also, PpsR2 controls the expression of PpsR1 which is a redox sensor and binds to a number of promoter regions within the major cluster of photosynthetic genes. There is therefore a complex interplay between light‐dependent antagonistic action of RpBphP1 on RpPpsR2 and redox potential on RpPpsR1 but not on RpPpsR2 Gene rpbphP1 was designed in silico to be used in spectroscopy, size exclusion chromatography and Ni2+ affinity column binding experiments. The gene was synthesized by GenScript  in plasmid pUC57 with NdeI restriction site at the 5′ end and sites HindIII and XhoI at the 3′. The gene was sub‐cloned, using STRU‐cloning protocol NdeI and HindIII), pET24a (NdeI and HindIII) and pET24a (NdeI and XhoI) to obtain expression constructs for N‐terminal His‐tag, C‐terminal His‐tag and untagged RpBphP1 protein respectively. For SAXS experiments gene rpbphP1 was PCR amplified from genomic DNA of bacterium Rhodopseudomonas palustris strain 2.1.6 and sub‐cloned into pET28a. Transformed E. coli BL21 (DE3) cells were grown to a density of OD600 = 0.6, induced with 0.02 mm IPTG and incubated overnight at 18 °C. Cells were harvested and re‐suspended in chromatography buffers containing 10 mm Biliverdin IXα , protease inhibitor tablets , DNaseI (10 μg·mL−1), Triton X‐100 (0.5%) and disrupted by French Press. Supernatants were loaded onto an affinity His‐Trap™ HP or ion exchange HiTrap QFF columns . Finally, proteins were gel filtered using a HiLoad 26/60 Superdex 200 column  in a 5 mm TrisHCl pH 8 and 10 mm NaCl buffer. Plasmid pET28a with incorporated gene ppsR2 from Rhodospeudomonas palustris was kindly provided by Shinji Masuda (Tokyo Institute of Technology) and Tom Beatty (University of British Columbia). Protein was expressed in E. coli BL21 (DE3), using the same protocol as RpBphP1, and purified on a His‐Trap HP column followed by gel filtration and stored in a final buffer of 20 mm Tris‐HCl pH 8, 300 mm NaCl, and 5 mm dithiothreitol.−2·s−1 at the sample. There was sufficient light to photo‐convert a Pfr dark state into a 100% Pr state within 1 min. The monochromatic light was passed through the top of the UV‐Vis cuvette, with a sample path‐length of 2 mm, and so full photo‐conversion to the Pr state was achieved over a wide range of protein concentrations, 0.04–4 mg·mL−1.UV‐Visible spectra were recorded at room temperature in a Perkin Elmer Lambda 35 UV/VIS spectrometer. Spectra were recorded in the dark or after illumination with monochromatic light produced by passing white light through an interference filter with a bandwidth of 10 nm centered on wavelength 760 nm . The fiber optic/filter system was placed ~ 4 cm from the sample which provided a photon flux density of ~ 250 μmol·m−1 in 5 mm TrisHCl pH 8 and 10 mm NaCl. For RpBphP1 this was either in the dark or after illumination with 760 nm light for 5 min. The column was calibrated with high molecular weight calibration kit . Data were plotted on a logarithmic scale using Kav, to obtain estimates of protein molecular weight in solution.Purified protein was loaded onto a Superose 12 10/300 GL in 200 μL volumes at concentrations between 1 and 4 mg·mL6‐tagged RpPpsR2 and untagged RpBphP1 or untagged RpBphP1ΔHOS was mixed in a ratio 1 : 4 respectively. Samples were pre‐incubated in the dark or illuminated for 5 min with light of wavelength 760 nm to achieve a full Pr state. Chromatography was carried out over 10‐20 min using a 1 mL His‐Trap HP column and an ÄKTA Explorer. The elution was monitored at the protein absorption wavelength 280 nm and the biliverdin IXα absorption a wavelength of 400 nm. RpBphP1 can only be eluted at high imidazole concentrations when in complex with His6‐RpPpsR2 and monitoring absorption at these two wavelengths allows peaks to be differentiated between those that contain His6‐RpPpsR2 only and those also in complex with RpBphP1.N‐terminal His−1 and 0.014 < S < 0.183 Å−1 respectively (Table m Tris buffer (pH 8) in a Vivaspin 500 centrifugal filter unit  to remove chemicals such as salt and imidazole. The sample was concentrated to ~ 4 mg·ml−1 and spin column flow through buffer was retained for SAXS data background subtraction. For the longer camera length, the sample was further diluted to 1 mg·ml−1. Samples were injected into a 50 μL volume brass cell with X‐ray path length of 1 mm covered on both sides by thin mica windows and chilled to 8 °C by water‐circulation around the brass cell. Data were collected as 60, 1‐min exposure, images on a time resolved multi‐wire area detector, assessed for time dependant radiation damage and averaged. A Pfr dark state was prepared by protein purification in the dark and pre‐incubation in the dark overnight. Pr state sample was induced by continuous illumination with white light passing through a 10 nm bandwidth interference filter centered on 760 nm  Ltd). The radius of gyration (Rg) was evaluated using the Guinier approximation I(S) = I(0)exp(−S2Rg2/3) for S..Rg < 1.3 and was also calculated with Dmax by the indirect Fourier‐transform program gnom. Molecular weights were determined with the program saxs mow2Small‐angle X‐ray scattering (SAXS) data were collected at STFC SRS facility beam line 2.1 at the wavelength λ = 1.54 Å. A SAXS X‐ray camera was assembled at two different lengths, 1.00 and 4.25 m, to cover scattering ranges 0.052 < S < 0.730 Åatsas (http://www.embl-hamburg.de/biosaxs/software.html) was used for data analysis. Ab‐initio envelopes of dark Pfr and illuminated Pr states were calculated from SAXS data in 10 runs, for each state, with the program damminsupcomb, damfilt and damaver were used to calculate average envelopes pymol equipped with the plugin CRYSOL to access the quality of model fit to SAXS data 2 as a measure of fit between calculated and measured SAXS scattering curves. A rigid body refinement program sasref was also used to perform an automated 3D search of domain alignments I(S)exp‐k.I(S)cal)/σ(S), as a function of S, where I(S)exp are measured intensities and I(S)cal are intensities calculated from atomic models, k a scale factor and σ(S) are SAXS data standard deviations The EMBL program suite RpBphP1 HOS and RpPpsR2 HTH domains were built so that full length models of RpBphP1 and RpPpsR2 could be created. Homolog proteins were found with the toolkit HHPredhttps://toolkit.tuebingen.mpg.de) and sequence aligned to their X‐ray structure sequences. Best homolog structures based on hit probabilities, usually 4 to 6 structures, were selected as PDB templates to automatically build 3D structures with the program modellerAtomic models of The authors declare no conflict of interest.ab‐initio envelope determination, modeling of SAXS data and measurement of UV‐Vis spectra. DB‐ cloning and sample purification for SEC measurements, Ni(II)‐affinity chromatography measurement of complex formation, critical reading of manuscript and measurement of UV‐Vis spectra. KE‐ PCR cloning of SAXS RpBphP1 sample, protein purification and characterization of purity by biochemical methods and UV‐Vis spectroscopy. GG‐ collection of SAXS dark and light induced state data, processing of raw images, reduction and analysis of SAXS data and revision of the manuscript. AF‐S‐ assessing quality of Pfr and Pr samples for SAXS experiments, checking SAXS sample purity by SDS/PAGE electrophoresis, UV‐Vis spectroscopy and assisting in SAXS data collection.MP‐ conception and design of experiments, writing and critical revision of the manuscript, SAXS data collection,


Herein, feasibility of supplementing wheat flour with Chinese yam powder (CYP) for noodle preparation was assessed. After supplementation with CYP, the alterations in chemical, texture, cooking, rheological, and microstructure attributes of noodles were observed. Due to higher protein and lower gluten, 20% of CYP promoted the stable network of gluten and starch particles. However, the excessive addition reduced the flexibility and the chewiness. The adverse changes were observed at 40% substitution level in texture profile analysis (TPA) and rheological parameters due to disrupted gluten–protein network which accelerated the exposure of starch particles. The CYP incorporation up to 20% showed better mouthfeel but further addition lowered the total sensory scores. Scanning Electronic Microscopy (SEM) analysis confirmed the modifications in noodles microstructure as CYP addition affected starch granule structure. In general, 30% substitution significantly improved the textural and rheological properties of noodles, indicating the potential of Chinese yam powder for industrial application. Noodles are rich in nutrition, easy to cook, and delicious traditional foods in Asian countries besides their increasing demand in many Western countries . TraditiIn the past years, researchers have been engaged to enrich noodles by fortifying them with various ingredients. The attempts have been made to use wheat noodles as a resource and then supplement them by adding broken rice, oat flour, barley flour, sweet potato flour, and buckwheat flour ,2,6,7. TDioscorea Polystachya (Turczaninow), is one of the important edible starch plants widely cultivated in China [Yam is recognized as a traditional staple food in many tropical regions in the world particularly in South Asia . Chinesein China  and it iin China  with genin China . Yam tubin China . Among tin China . Previous studies were conducted to evaluate the functional properties of noodles and the effects of various compounds to improve their final quality . Noodle Wheat flour  and table salt were purchased from Carrefour supermarket in Hefei city, China. The dried Chinese yam thin piece was procured from Tanan Local Wholesale Shop . At the laboratory, Chinese yam powder (CYP) was ground using Xichu Food Grinder , and passed through 100 mesh sieve.The proximate composition  of purchased wheat flour and CYP were tested by using the standard methods of Association of official analytical chemists (AOAC) . Ash conw/w). The aluminum crucible was stored under refrigerated conditions (4 °C) for 12 h. The empty crucible was taken as control. Initially, the starting temperature of the sample was 30 °C. After holding at 30 °C for 1 min, sample was heated to 90 °C at a rate of 10 °C/min [The starch gelatinization properties of wheat flour supplemented with different concentrations  of CYP were determined by Differential Scanning Calorimetry (DSC) . Briefly0 °C/min . The folTp: gelatinization temperature peak; To: onset gelatinization temperature; Tc: conclusion gelatinization temperature; R: gelatinization temperature range. R was calculated following Formula (1).According to a previous study with few modifications , the far−1 at 20 °C and as a function of frequency (0.1 to 10 Hz) using a Discovery Hybrid Rheometer-3  equipped with a cone-and-plate geometry . All parameters were tested as per the above-mentioned experimental conditions.Rheological evaluation, especially in the linear viscoelastic region, has been used for unraveling the structure and properties of dough in order to study the functions of dough ingredients . Elasticw/w)-supplemented wheat flours mixed with 50% distilled water, 2% NaCl, and 0.1% NaHCO3. To prepare the dough, 100 g of supplemented powder (different yam levels) was mixed with required amount of water in an automatic noodle machine . The dough was sheeted to about 2 mm thickness and the resulting fresh noodles were cut into 15 cm.Based on an earlier study , noodles1) and put into a beaker with 400 mL of boiled water. After cooking for 2 min, the noodles were allowed to cool down by placing them in cold water for 30 s followed by drying for 3 min and weighed (W2). The cooking yield of the noodles was calculated using the following Formula (2)For this, noodles (15 cm) strips were cooked for 2 min. According to the method of Lu et al. (2009) with few modifications , cooking2 = Weight of cooked noodle after drying; W1 = Weight of raw noodle.W3) is recorded as the weight of beaker after drying. The cooking loss of the noodles was calculated using the following Formula (3)For the cooking loss, the final weight  , three nFor sensory evaluation, five types of noodles samples (100 g) were cut into 15 cm length and cooked up to their optimal time (the time when white core in the cross section disappeared) in 100 °C water. The samples were dried for not more than 10 min in tightly covered plastic food containers before testing. Sample noodles were evaluated by 20 students and staff of the Food Science and Engineering College, Hefei University of Technology. All the samples were coded in a randomized order and evaluated by a 9-point hedonic scale . The different sensory attributes included color, appearance, taste, flavor, hardness, toughness, and overall acceptability. The raw and cooked samples were cut into 3 cm and frozen at −80 °C. After freeze-drying, the dried samples were cut into cross-section and placed on a specimen holder for coating with gold for 90 s. The microstructure of samples was observed by Scanning Electron Microscope , at 22 KV with 250× and 1000× magnification.All the measurements were performed in triplicate and the obtained results were expressed as the mean values ± standard deviation (SD). The results were statistically analyzed using SPSS . For the data analysis, one-way analysis of variance (ANOVA) was performed using the Origin Lab  software.The chemical compositions of original wheat flour, CYP, and the mixed flour with different amounts of CYP was presented in p < 0.05) as compared to CYP. From At first, lower gelatinization peak temperature in wheat flour was observed . The addition of CYP decreased the springiness of wheat flour noodles which might be due to decreased gluten E. No gluThe hardness initially increased from 25,504.35 g to 26,440.21 g after the addition of CYP (up to 30%) F which mIt can be seen from the The CYP-fortified cooked noodles were evaluated for their sensory properties and the results are presented in Up to 20% concentration of CYP, some sensory properties were satisfactory such as hardness, mouthfeel, and springiness, which was also revealed in TPA and SEM properties. Incorporation of more than 20% of CYP resulted in low total score and the noodles were not acceptable due to diluted gluten. Similarly, in an earlier study by Sanju and Kawaljit [The SEM microstructures of raw noodles and cooked noodles are shown in Our study explored the possibility of preparing noodles with acceptable quality from wheat flour supplemented with different levels of Chinese yam powder. Rheological, farinograph, and gelatinization texture properties were evaluated. It can be concluded that the addition of CYP to wheat flour can affect the overall properties of dough and noodles. When supplemented, the texture of noodles as evidenced by several parameters such as water absorption, cooking loss and firmness were improved. For sensory evaluation, some properties such as hardness, mouthfeel, and springiness were improved up to 20% incorporation of CYP, which supported the TPA properties. For microstructure, SEM revealed that addition of CYP could promote the close binding of starch particles to gluten protein and thereby resulted in relatively smooth surface. The above results encourage further studies to confirm the beneficial effects in terms of improved nutritional values of the noodles prepared from wheat flour supplemented with acceptable levels of CYP. They can in fact be advantageous for consumers seeking alternatives containing natural healthy ingredients in foods. The results of the current study demonstrate the potential for extending the use of Chinese yam powder in wheat noodle for industrial application.


Lck/Yes related novel protein tyrosine kinase) belongs to the family of Src-related non-receptor tyrosine kinases. Consistent with physiological roles in cell growth and proliferation, aberrant function of Lyn is associated with various forms of cancer, including leukemia, breast cancer and melanoma. Here, we determine a 1.3 Å resolution crystal structure of the polyproline-binding SH3 regulatory domain of human Lyn kinase, which adopts a five-stranded β-barrel fold. Mapping of cancer-associated point mutations onto this structure reveals that these amino acid substitutions are distributed throughout the SH3 domain and may affect Lyn kinase function distinctly.Lyn kinase ( Lck/Yes related novel protein tyrosine kinase) is a Src-family kinase organized into four domains. These include an N-terminal unique domain , two adapter domains (SH3 and SH2) and C-terminal kinase domain . Src-family kinase activity is conformationally regulated via distinct interactions between these domains. In the inactive state, the SH3 domain binds to the linker between the SH2 domain and the kinase domain, keeping the kinase in a closed (inactive) conformation [Lyn kinase  cells were transformed with pDONR223-Lyn (Addgene plasmid # 23905) containing the coding sequence of SH3 domain of Lyn kinase in pET24(+) vector (Novagen). Cells were grown at 37°C in 1 L LB medium containing 100 mg/L ampicillin. At an OD600 of 0.8, expression was induced with 1 mM IPTG and the temperature was lowered from 37°C to 30°C. After 5 h, cells were harvested at 9,180 × g, and the pellet was resuspended in 50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA and 50 mM NaCl (25 ml per 1 L of culture). Protease inhibitor cocktail , 2 mM MgCl2 and DNase 1 (20 U per 1 L of cell culture), 1 mM TCEP, and 100 mg/L lysozyme were added to the suspension. The suspension was sonicated  and the lysate was centrifuged for 1h at 4°C at 38,360 × g. The supernatant was passed through a 0.45 μm filter, and loaded on a 5 ml His-Trap HP column equilibrated in wash buffer . The protein was eluted in wash buffer containing 250 mM imidazole. Protein was further purified by size exclusion chromatography on a Superdex 200 10/300 GL column equilibrated with 20 mM Tris-HCl, pH 7.5, 5% glycerol, 150 mM NaCl, and 1 mM TCEP.Table 1).Crystals of purified Lyn SH3 domain grew by the hanging drop vapor diffusion method using 1.5 μl of Lyn SH3 domain (10 mg/ml in 20 mM Tris pH 7.5) equilibrated with 1.5 μl of reservoir solution  and appeared within 24 h at 22°C. Crystals were cryo-protected using 0.09 M Na citrate pH 3.5, 2.88 M NaCl and 10% glycerol, and were flash cooled by plunging in liquid nitrogen. Data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) Experimental Station 9–2 using a Pilatus 6M detector (Table 1) and the structure determined in the Phaser subroutine [Table 1). The model of the C-terminus focused on the conformation with better electron density but could not be modeled with accuracy, and contains two rotameric outliers. Structural superpositions were performed in Coot [Data were processed using HKL2000   contains five β-strands organized into two β-sheets. Previously termed β1 –β5, these β-strands are connected via four loops termed the RT loop, n-src loop, distal loop, and 310 helical loop. Comparison of our crystal structure with the structure of the unliganded Lyn SH3 domain determined by NMR spectroscopy (PDB 1WIF) [Fig 2A). The largest differences in position involve the residues that are found within the polyproline binding cleft, G76, H78 and P79 of the RT loop and E98, E99 of n-src loop, which exhibit positional differences of 1.5 Å – 2.3 Å due to rotation of these loop elements. Conformational changes in these two loops are observed in the NMR structure bound to the herpesvirus-derived peptide [Fig 2B and 2C).The crystal structure of the Lyn SH3 domain (DB 1WIF)  yields a peptide  . While cell assays evaluating the impact of these single amino acid changes have not, to date, been reported, the use of computational prediction algorithms suggests that 12 of these substitutions are likely to be transformative and contribute to disease.Advances in sequencing now allow mutations in individual cancer patients to be identified. Analysis of whole genomes revealed 18 cancer-associated point mutations within the Lyn SH3 domain, affecting ~30% of the residues in this domain –25 . For example, a subset of the mutations, including D81N, W99L and E98K, may disrupt the protein-protein interaction interface with the kinase domain, and eliminate negative regulation of kinase activity. Other mutations, such as V118M, may destabilize the fold of the SH3 domain, again preventing the normal attenuation of kinase activity in the basal state of Lyn. In contrast, single amino acid substitutions that are predicted to be non-transformative are either relatively conservative, or are located on the surface of the SH3 domain that is not engaged by the kinase domain.Evaluation of the locations of these mutations in the context of the structure suggests that many of the likely transformative substitutions could prevent the interaction of the SH3 domain with the kinase domain, but by distinct mechanisms (The crystal structure of the Lyn kinase SH3 domain improves the molecular understanding of the regulatory mechanism of the Src family kinases. The mapped cancer-associated mutations in the SH3 domain identify how different regions of this domain affect protein function.


A coupled macro-meso-scale numerical simulation is applied to investigate the friction effect on texture evolution during uniaxial compression of NiTi shape memory alloy at 400 °C. In this approach, macroscale finite element simulations in consideration of various friction coefficient are conducted and then the corresponding velocity gradients in various regions are extracted mainly based on the delivery deformation gradient in the user-defined material subroutine (UMAT) in ABAQUS code. These velocity gradients are regarded as the deformation conditions applied in the mesoscale VPSC model. Simulation results in terms of macroscale finite element modeling demonstrate that only within the region of minimum deformation zone which is close to the die, friction effect has a nonnegligible influence on the velocity gradient. Simulation results with respect to the mesoscale VPSC modeling show that the affine and Neff = 10 linearization schemes provide the best predictions for NiTi shape memory alloy with cubic structure. Furthermore, the friction effect does have an influence on the evolution of slip mode activities in various deformation zones and therefore results in the inhomogeneous texture evolution within the deformed sample during uniaxial compression. The frictional behavior between a workpiece and forming tool is unavoidable during the real-world plastic deformation conditions, as Tabor in 1951 had proposed that there still exists a measurable coefficient of friction ranging from 0.1 to 0.15 between highly polished metallic surfaces and diamond . Numerous experimental methods and simulation tools have been proposed to determine such friction effect on the texture evolution of metallic materials during various plastic deformation process, including nano-indentation , cold-roTo date, there still exists certain restrictions in terms of revealing the plastic deformation mechanisms of metallic materials under simple deformation conditions, including uniaxial compression/tension, simple shear, and plane strain compression, via experimental measurements . TherefoIn the present study, texture evolution of NiTi shape memory alloy undergoing uniaxial compression at 400 °C is investigated in consideration of the friction effect via VPSC model. Firstly, the needed material parameters for NiTi shape memory alloy are calibrated by means of the particle swarm optimization (PSO) algorithm . SubsequIn the present study, texture evolution during uniaxial compression of NiTi shape memory alloy at 400 °C is simulated via VPSC model, which is developed by Lebensohn et al. . VPSC st−1 in consideration of experimental loading rate. The parameter m qualifies the strain rate sensitivity of material and its value is chosen to be 20 indicating a certain rate sensitivity of NiTi shape memory alloy [For the slip-based deformation, the shear rate ry alloy . sgn(.) The CRSS in each slip system α evolves by the following extended Voce law.–1. It is important to note that a heating device is applied to heat both the specimen and dies to 400 °C before loading. In other words, NiTi shape memory alloy specimen is always to be subjected to compression deformation at a constant temperature of 400 °C. This operation is to confirm the isothermal condition during uniaxial compression. Then the measured stress-strain curve shall not only be used to fit the material parameters in the VPSC model at the mesoscale but also used to obtain the elastic and plastic data which are needed during the finite element modeling at the macroscale. In addition, the applied grain orientations in the VPSC model are derived from the measured texture of the adopted NiTi shape memory alloy using the electron back-scattered diffraction (EBSD) equipment, as shown in To simulate uniaxial compression along the normal direction with VPSC model, the component of velocity gradient Chapius et al.  have conBased on the aforementioned PSO algorithm, the optimized material parameters are obtained and shown in Based on the identified material parameters as shown in Although these simple deformation conditions can be easily conducted in the VPSC simulations in terms of the velocity or displacement gradients, including uniaxial compression/tension, plane strain compression and simple shear, the deformation history in consideration of the friction effect is rather complicated and would be implemented with difficulty in the simulations using VPSC model. Hence, finite element modeling counting on the friction effect during plastic deformation is needed to extract the deformation history in some selected elements. In the present study, the method proposed by Li et al.  and LatyIn continuum mechanics, the velocity gradient tensor By using a Taylor expansion, the total deformation gradient The aforementioned equations in terms of calculating the velocity gradient can be readily implemented in the user-defined material subroutine (UMAT) in ABAQUS code, in which ABAQUS code passes ent step . −1 in consideration of friction effect, as shown in As for finite element modeling of uniaxial compression at a constant strain rate of 0.001 sDue to the aforementioned investigations, it can be concluded that the friction influences the velocity gradient within the deformed sample and further contributes to the texture inhomogeneity during plastic deformation. It is well known that the deformation mechanisms have a significant influence on the texture evolution of the deformed sample. Therefore, it is of great importance to investigate the deformation mechanisms during uniaxial compression of NiTi shape memory alloy in consideration of friction effect. The slip mode activities with respect to {110} <100> slip mode, {110} <111> slip mode and {010} <100> slip mode during uniaxial compression at the deformation degree of 20% are all shown in By using the PSO algorithm in parameter calibration, the material parameters used in VPSC model are identified with efficiency and accuracy. In addition, among the accessible linearization schemes in VPSC model, the affine scheme and Neff = 10 scheme give the best predictions.The friction effect has an obvious influence on the plastic deformation in the minimum deformation zone close to the dies and further affects the corresponding texture evolution during uniaxial compression. In the intermediate deformation zone and the principal deformation zone, the friction effect on the velocity gradient rapidly decrease and therefore has a minor influence on the texture evolution.In the minimum deformation zone close to the dies, the friction coefficient not only affects the evolution laws with respect to the slip mode activities but also the suffered plastic strain. Whereas in the intermediate deformation zone and the principal deformation zone, there exist relatively similar evolution laws with respect to the slip mode activities, but the evolution rates with respect to {110} <100> slip mode and {110} <111> slip mode are fastest in the principal deformation zone, which contributes to the change of intensity with respect to <111> fiber texture component.In the present study, the coupled finite element modeling via ABAQUS code and crystal plasticity modeling via VPSC model are applied to shed a light on the friction effect on texture evolution during uniaxial compression of NiTi shape memory alloy with the assistance of dislocation slip. The following conclusions can be drawn.


Saccharopolyspora erythraea that dimethylates A2058 in 23S rRNA using S-adenosyl methionine (SAM) as methyl donor, protecting the ribosomes from macrolide binding. To gain insights into the mechanism of macrolide resistance, the crystal structure of ErmE was determined to 1.75 Å resolution. ErmE consists of an N-terminal Rossmann-like α/ß catalytic domain and a C-terminal helical domain. Comparison with ErmC’ that despite only 24% sequence identity has the same function, reveals highly similar catalytic domains. Accordingly, superposition with the catalytic domain of ErmC’ in complex with SAM suggests that the cofactor binding site is conserved. The two structures mainly differ in the C-terminal domain, which in ErmE contains a longer loop harboring an additional 310 helix that interacts with the catalytic domain to stabilize the tertiary structure. Notably, ErmE also differs from ErmC’ by having long disordered extensions at its N- and C-termini. A C-terminal disordered region rich in arginine and glycine is also a present in two other MTases, PikR1 and PikR2, which share about 30% sequence identity with ErmE and methylate the same nucleotide in 23S rRNA.Pathogens often receive antibiotic resistance genes through horizontal gene transfer from bacteria that produce natural antibiotics. ErmE is a methyltransferase (MTase) from  Macrolide antibiotics and their third-generation ketolide derivatives are used against a broad range of Gram-positive pathogens. They inhibit translation by binding in the nascent peptide exit tunnel close to the peptidyl transferase center of the large ribosomal subunit5. One important mechanism of microbial resistance to macrolides is the N6 methylation of 23S rRNA nucleotide A2058 (Escherichia coli numbering) in the macrolide binding site by the Erm (erythromycin ribosome methylation) group of MTases6. This group of enzymes uses SAM to specifically mono- or dimethylate a 50S ribosomal precursor substrate8, where A2058 is accessible for modification. Erm genes were originally identified in microorganisms producing natural macrolides as a mechanism of self-protection against their own antibiotics9. ErmE (EC 2.1.1.184) is a dimethyltransferase from the actinomycete Saccharopolyspora erythraea, from which the first macrolide antibiotic erythromycin was originally extracted11. ErmE provides resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics to S. erythraea12. However, in conditions of wide use of antibiotics, horizontal gene transfer has led to propagation of pathogens carrying this and other erm genes.The ribosome is the large macromolecular machine responsible for the sequential template-dependent polymerization of amino acids into a polypeptide chain13 and present a very promising class of antibiotics14. Most members of this class are synthetic and semi-synthetic derivatives of macrolides. However, Streptomyces venezuelae strain ATCC 15439 produces the natural ketolide pikromycin15. To avoid self-inhibition, this microorganism expresses two MTases PikR1 and PikR2 that mono- and dimethylate A205816, the same nucleotide as ErmE. PikR1 and PikR2 display 39% sequence identity to each other and 35% and 33% sequence identity to ErmE.Ketolides were initially developed to overcome macrolide resistanceIn light of the danger of horizontal transfer of macrolide and ketolide resistance genes, there is an urgent need for better understanding of the respective resistance mechanisms, including information on the structural and functional properties of ribosome-modifying enzymes. Here, we present the crystal structure of rRNA MTase ErmE, and analyze the similarities and differences to PikR1, PikR2, ErmC’ and other similar MTases.8-tagged constructs were synthesized by GenScript and subcloned into the pET-24a(+) vector . Cultures were grown at 37 °C in LB media with 0.025 mg/ml kanamycin and 0.1% (w/v) D-glucose until an OD600 of 0.6. Protein expression was induced with 0.1% (w/v) L-arabinose. After overnight cultivation at 18 °C, the cells were collected by centrifugation, resuspended in lysis buffer  supplemented with 10 mM imidazole, 10% (v/v) glycerol, 0.06 mg/ml DNAse and cOmplete protease inhibitor cocktail , and lysed in a high-pressure homogenizer . The lysate was centrifuged for 1 h at 30,000 g and the supernatant was applied to a gravity-flow column containing Ni-sepharose resin  equilibrated with lysis buffer and 10% (v/v) glycerol. The column was washed with lysis buffer containing 20 and 30 mM imidazole and protein elution was performed with 500 mM imidazole in 50 mM phosphate buffer pH 8, 0.3 M NaCl and 2 mM ß-mercaptoethanol. Eluted protein was dialysed against 20 mM Tris-SO4 pH 8, 0.8 M (NH4)2SO4, 2 mM ß-mercaptoethanol and loaded on a 5 ml HiTrap Phenyl HP column  equilibrated with dialysis buffer. Elution was done with a linear gradient of (NH4)2SO4 (0.8–0 mM) in 20 mM Tris-SO4 pH 8. Size-exclusion chromatography (SEC) was performed using a HiLoad 16/600 Superdex 75 pg column  equilibrated with running buffer 2SO4 and 2 mM ß-mercaptoethanol). Peak fractions were analysed with SDS-PAGE and concentrated to 10 mg/ml using a 10 kDa cutoff Vivaspin Turbo concentrator . Purification was performed at 4 °C.Plasmids were transformed into 17 was done using a BioRad CFx connect real time PCR machine.Differential scanning fluorimetry (DSF)18 of the European Synchrotron Radiation Facility , the crystal was fished directly from the drop and flash frozen in liquid nitrogen. X-ray experiments were done at 0.9677 Å wavelength at 100 K.All proteins were subjected to sitting drop vapor diffusion crystallization using a mosquito crystallization robot . Rhomboid-shaped tetragonal crystals of truncated ErmE grew in 5 d at room temperature in drops of 200 nl in 2% (v/v) tacsimate pH 5.0, 0.1 M sodium citrate tribasic dihydrate pH 5.6 and 16% (w/v) PEG 3350 . For data collection at beamline ID30A-3 (MASSIF-3)XDS19. The structure was solved by molecular replacement with Phaser20, using as search model an ensemble generated from PDB IDs 1QAM21, 3FUU22, 1YUB23, 3FYC24 and 1ZQ9 by CCP4 online pipeline MrBump25. The structure was traced with PHENIX AutoBuild26 followed by completion of missing regions in ARP/wARP27. Manual rebuilding was done in Coot28 and refinement with phenix.refine29. Protein geometry was validated in MolProbity30. All figures representing structures were made using PyMOL31.Data was processed using 2mFo-Dfc map is presented in Supplementary Fig. Data collection and refinement statistics are reported in Table 4 was used after IMAC, to avoid formation of salt crystals during crystallization.After initial purification tests, DSF measurements showed that the thermal stability of PikR1 increased in presence of phosphate and sulphate. For this reason, phosphate buffer was used during lysis and Tris-SO8-tagged PikR1, we hypothesized that it was the result of a C-terminal proteolytic degradation. In support of this, investigation of the PikR1 sequence with the PrDOS online server32 predicted disorder of a C-terminal region of around 67 aa, which could make the protein susceptible to proteolytic degradation as well as prevent crystallization of the full-length protein. For PikR2 and ErmE, C-terminal regions of 64 and 93 residues were similarly predicted to be disordered  including residues 1–290  from Bacillus subtilis (PDB ID 1QAM)21 as the structure most similar to ErmE, with root mean square deviation (rmsd) of 2.75 Å over 230 Cα atoms of the superposed structures 22 and its archaeal homologue Dim1 24. Since these enzymes modify a different RNA substrate, we decided to mainly compare the ErmE structure to the structure of ErmC’.The bilobed structure of ErmE consists of an N-terminal Rossmann-like α/ß catalytic domain (residues 42–211) and C-terminal helical domain (residues 219–285), which are connected by a loop Fig. . DALI34 res Fig. . Interes35, and the most common fold of the catalytic domain of SAM-dependent MTases36. The C-terminal domain was in ErmC’ proposed as an RNA-binding domain37 based on its large positively charged surface. However, it was later shown by mutagenesis that the key residues for specific RNA binding are located in the catalytic domain, facing the cleft between the domains. Accordingly, the C-terminal domain was suggested to mainly function in structural stabilization of the catalytic domain38.The Rossmann-like fold is common for nucleotide-binding proteins in general10 helix (η3) on one side, and three α-helices (α4-α6) and two 310 helices (η1-η2) on the opposite side  and two antiparallel (ß0 and ß7) ß-strands that are surrounded by three α-helices α1-α3) and one 3ices η1-η on the o and one α atoms, the main difference being a longer loop between helices α5 and α6 in ErmE.ErmE and ErmC’ have the same topology of their catalytic domains Fig.  that shaThe N-terminal domain shows an L-shaped pocket rich in conserved residues Figs  and 5. C3DLigandSite server39 predicts the same binding site for SAM. Most SAM-binding residues are conserved from ErmC’, with minor differences presented by Ile13/Leu44 and Ser39/Pro70, involved in main-chain interactions with SAM, and Ile85/Phe116, where the side chain makes a hydrophobic interaction with the adenine group of SAM . Intriguingly, PikR1 that was reported to be a monomethylating MTase16, instead of the (N/D)PP(Y/F/W) motif associated with monomethylating N6A-MTases, contains the same NVPF motif as the dimethyltransferase PikR2 .These residues are part of the sequence motif IVmC’ Fig. , that is10 helix (η4)  and one 3η4) Figs  and 3. Tses Fig. . Anotherses Fig. .Together with the C-terminal loop, η4 participates in interactions with the loop between α5 and α6 of the catalytic domain, contributing to stabilising the structure of ErmE Fig. . A hydroIn addition, the difference in interactions between N- and C-terminal domains in ErmE and ErmC’ leads to the slightly different orientation of these domains relative to each other, which results in a higher rmsd value for the superposition of the whole MTase structures as compared to when the individual domains are superposed.43 and RlmJ44 and catalytic domains of the human mRNA N6A-MTases METTL3-METTL1445 and METTL1648. These enzymes all display similar structures of their catalytic domains but make use of a variety of loops, tails or extra domains for specific recognition of the sequence and structure of their respective RNA substrates. There are few structures of N6A MTases in complex with RNA; KsgA50 and METTL1647. KsgA methylates a close to mature 30S subunit and similarly to the Erm family of MTases contains a C-terminal helical domain. For ErmE, it has been shown that although the natural substrate is a precursor of the 50S ribosomal subunit, the enzyme can specifically methylate a 27-nucleotide stem loop RNA substrate mimicking the local environment of A205851. Thus, some essential recognition elements in the RNA are located in close proximity to the adenosine that is methylated.In addition to ErmE, ErmC’ and ErmAM that provide antibiotic resistance, structures are available of two bacterial rRNA N6A-MTases involved in ribosome biogenesis, KsgA38. In the ErmE structure, a sulfate ion is bound between Lys164 and Arg174, possibly mimicking a substrate phosphate. The positively charged surface of the C-terminal domain is also likely to contribute to substrate binding.On the protein side, mutational studies on ErmC’ showed that a single arginine in equivalent position to Lys164 in α5 of ErmE is essential for erythromycin resistanceThe N-terminal disordered region of ErmE is rich in arginine, while the C-terminal disordered region is dominated by glycine and arginine. Predicted disordered low-complexity regions with similar characteristics are also present in PikR1 and PikR2. These regions may contribute to binding of the 50S ribosome assembly intermediate where A2058 is accessible for modification. Similarly to the positively charged tails of ribosomal proteins, they may order upon interaction with the negatively charged RNA backbone. However, ErmC’ does not contain the corresponding long tails but can still recognize and modify the same substrate (Fig. 52. The strategy was based on exchanging the C-terminal domain, the N-terminal tail including α1 and η1 and the loop between α7 and α8 in KsgA to the corresponding sequences from ErmC’. The structure-guided sequence alignment of ErmE with ErmC’, ErmAM and KsgA (Fig. Recently, KsgA was engineered to alter its substrate specificity and allow activity on the Erm substrateHere, we present the first crystal structure of rRNA methyltransferase ErmE, determined at 1.75-Å resolution. The structure of the enzyme could be potentially used for structure-based drug design with the aim to prevent macrolide antibiotic resistance in pathogens. Considering its higher than 30% sequence identity to PikR1 and PikR2, the structure of ErmE is also expected to be useful as a molecular replacement search model for further studies of PikR MTases.Supplemental material


Highest protein content of 28.87% was recorded in blend with 20% groundnut protein concentrate. Fat, ash, and crude fiber contents ranged from 1.93% to 8.84%, 0.70% to 1.13%, and 0.84% to 1.23%, respectively. Carbohydrate decreased significantly (p < .05) with increasing concentration of groundnut protein concentrate. Bulk density and swelling capacity of the flour blends reduced significantly (p < .05) with increasing level of groundnut protein concentrate flour while solubility index, water absorption, emulsion, and foaming capacities increased. Peak time, peak, trough, breakdown, final, and setback viscosities of the flour blends reduced with groundnut protein concentrate inclusion while the pasting temperature reduced. Overall, wheat/groundnut protein concentrate flour blends showed good functional and pasting properties.This study investigated the effect of groundnut protein concentrate inclusion on the quality of wheat flour. Wheat and groundnut concentrate flours were blended  at ratios 100:0, 95:5, 90:10, 85:15, and 80:20, with 100% wheat flour serving as the control sample. Subsequently, proximate composition, functional, and pasting properties of blends were determined using established methods. Protein content of the concentrate was 72.80%. Significant ( The resulting pellet was air‐dried at ambient temperature (32±2°C) for 48 hr to rid it of solvent odor. It was then milled and packed in a plastic container  and stored in a cool place (10 ± 2°C) until required for use.Defatted groundnut flour was produced based on the procedure described by Kudre, Benjakul, and Kishimura  with lit2.2.2Groundnut protein concentrate was produced using the procedure reported by Gayol et al. . Defatte2.2.3Wheat flour and GPC were blended  at ratios 100:0, 95:5, 90:10, 85:15, and 80:20, with 100% wheat flour serving as the control sample. The samples were homogenized with the aid of a Kenwood Mixer to obtain homogenous samples.2.2.4Protein, fat, ash, moisture, carbohydrate, fiber contents, and energy values were determined using standard methods AOAC, . Bulk dePasting characteristics of blends were evaluated using a Brabender viscoamylograph . Flour slurry, containing 12% solids, was heated from 30 to 95°C at a rate of 2.5°C/min, held at 95°C for 15 min, and cooled at the same rate to 50°C  version 23.0).33.1Proximate composition of GPC is presented in Figure 3.2p < .05) reduction in moisture content. This is advantageous because reduction in moisture content will reduce the proliferation of spoilage organisms especially mold, thus, improving the shelf stability of the product.Proximate composition of blends is presented in Table p < .05) increase in protein content of wheat flour was recorded with increasing GPC. Highest protein content of 28.87% was recorded in blend with 20% GPC. Vegetable proteins in forms of flour, concentrates, and isolates have been incorporated in many food systems for better nutritional values and functionality than products produced solely from wheat flour  with increasing GPC. The increase in protein on the one hand, and the decrease in carbohydrate on the other hand, was a direct consequence of increasing and decreasing GPC and wheat flour, respectively.Significant  with increasing GPC. Lower bulk density in the blends compared with 100% wheat flour could be a result of reduction in carbohydrate content which has been reported to have high bulk density  effect on water absorption capacity; however, there was a significant (p < .05) increase in water absorption capacity in blends with 10% groundnut concentrate and above. The increase in water absorption capacity could be due to high water absorption capacity of GPC which probably improved the structural matrix for holding water, sugars, and other components  increase in emulsion capacity. This may be due to the presence or increase in soluble proteins due to inclusion of GPC. According to Garba and Kaur  with increasing GPC in the blends probably due to the increased protein content of the blends. Brou et al.  increase in solubility index. This could be due to the high concentration of soluble protein in groundnut protein concentrate  from 1,492 to 913 RVU with increasing GPC. Reduction in peak viscosity could be due to a lowering of the starch as well as interactions between the starch, fat, and protein contents of the blends. Peak viscosity has been reported to be correlated with water binding capacity of starch which takes place at equilibrium point between swelling which causes an increase in viscosity while rupturing and realignment cause its reduction  difference between 100% wheat flour and blend containing 95% wheat flour and 5% GPC in pasting temperature. However, significant (p < .05) increase in pasting temperature was observed at concentration of 10% groundnut protein concentrate and above. Generally, the pasting properties (except pasting temperature) and the proximate composition of the flours indicate a direct relationship between the starch (carbohydrate) content and the pasting properties on the one hand, and an inverse relationship between protein and the pasting properties on the other hand. As the carbohydrate decreased, the pasting attributes also decreased, but as the protein increased, the pasting attributes decreased. The decrease in pasting attributes (except pasting temperature) with increasing protein may be due to the effect of protein on the starch. Chandrashekar and Kirleis  with increasing level of GPC while solubility index, water absorption, emulsion, and foaming capacities increased. Peak time, peak, trough, breakdown, final, and setback viscosities of the flour blends reduced with GPC inclusion while the pasting temperature increased. Overall, wheat/groundnut protein concentrate flour blends showed good functional and pasting properties indicating that the blends can be used in the production of bread, cakes, biscuits, pancakes, and other baked products.This study showed the effect of the different levels of GPC in wheat flour on the proximate, functional, and pasting characteristics of the flour blends. The protein, fat, ash, and crude fiber contents of the blends improved with the inclusion of GPC while the moisture and carbohydrates reduced. Bulk density and swelling capacity of the flour blends reduced significantly (None declared.On behalf of all coauthors, I, Dr. Ocheme Boniface Ocheme, declare that this article has not been published in part or whole elsewhere. Neither is it under consideration for publication in another journal. All authors were actively involved in the work leading to the manuscript and will hold themselves jointly and individually responsible for its content.