GT), have helped define the role of the αIIbβ3 integrin in platelet aggregation. Stable bent αIIbβ3 undergoes conformation changes on activation allowing fibrinogen binding and its taking an extended form. The αIIb genu assures the fulcrum of the bent state. Our goal was to determine how structural changes induced by missense mutations in the αIIb genu define GT phenotype.Studies on the inherited bleeding disorder, Glanzmann thrombasthenia (ITGA2B and ITGB3 in the index case followed by in silico modeling of all known GT‐causing missense mutations extending from the lower part of the β‐propeller, and through the thigh and upper calf‐1 domains.Sanger sequencing of ITGA2B coding for a p.Asp591Ala substitution in an interconnecting loop of the lower thigh domain of αIIb in a patient with platelets lacking αIIbβ3 led us to extend our in silico modeling to all 16 published disease‐causing missense variants potentially affecting the αIIb genu. Modifications of structuring H‐bonding were the major cause in the thigh domain although one mutation gave mRNA decay. In contrast, short‐range changes induced in calf‐1 appeared minor suggesting long‐range effects. All result in severe to total loss of αIIbβ3 in platelets. The absence of mutations within a key Ca2+‐binding loop in the genu led us to scan public databases; three potential single allele variants giving major structural changes were identiffied suggesting that this key region is not protected from genetic variation.A homozygous c.1772A>C transversion in exon 18 of IIb genu is the object of stringent quality control to prevent platelets from circulating with activated and extended integrin.It appears that the α Clinical and biological data were obtained retrospectively from the cited publications and are summarized and referenced in Table http://www.exac.broadinstitute.org) and Ensembl genome browsers . For amino acid numbering, +1 starts with the initiating Met with signal peptide included. This is often followed by that of the mature protein (in parentheses), this involves subtracting 31 amino acids from the HGVS numbering of αIIb and 26 for β3. Amino acid numbering for the mature protein was previously used in the crystal structure of αIIbβ3 and subsequent in silico modeling  nomenclature for cDNA and protein is used throughout unless stated otherwise. For nucleotide numbering, the A nucleotide of the ATG start codon was designated +1 (cDNA 2.1.4http://www.pymol.org) and 3fcs or 1uv9 pdb files for crystal structures of αIIbβ3 as described by us  G = 0.000008354 (ExAC). This variant gives a p.Asp591Ala substitution (D560A for mature αIIb). Asp591 is highly conserved within vertebrates and is retained in all human integrin α‐subunits  affecting exon 18 of its Figs  and S2. 3.2ITGA2B and ITGB3 identiffied by whole exome and whole genome human sequencing in over 16,000 normal individuals in the ThromboGenomics project. Nevertheless 16 (thigh) and 23  nonsynonymous missense variants of unknown pathogenicity were present. A current assessment of the aa471‐769 sequence in the ExAC database  revealed 97 missense variants. This increased to 134 when Ensembl was consulted; a list that now includes presumed heterozygous expression of eight of the 15 variants causative of GT when homozygous or part of compound heterozygosity. Clearly, the genu and surrounding regions of αIIb are undergoing constant evolutionary genetic change.A comprehensive search of the literature revealed 15 missense variants causative of GT within the aa471‐769 sequence that begins with the C‐terminal strands of the β‐propeller, extends through the thigh domain and terminates with the upper domain of calf‐1 Table  Figures. All wer3.3To better understand the pathological influence of the Asp591Ala variant we first performed in silico modeling of domains close to and including the upper connecting loops of the genu.C‐terminal domain of the β‐propeller. The only known disease‐causing β‐propeller mutation in our selected aa471‐769 sequence is a homozygous Ala477Pro (A446P) substitution in a Chinese boy with type II GT  is with Arg551 of αIIb  has been the object of several reports mostly from Europe, giving rise to type I GT with moderate to severe bleeding Table . Two casA homozygous Pro507Arg (P476R) thigh domain substitution was detected in a patient from India  involved in a disulfide bridge and four amino acids engaging in Ca2+ coordination   Figure b. At the) Figure b: the fiNonsynonymous variants in the thigh domain associated with mRNA loss. Homozygous Gln626His (Q595H) giving type I GT was independently detected in four European studies suggesting a possible founder effect (Jallu et al., 3.42‐terminus of the thigh domain through interconnecting loops. Our literature research identiffied nine disease‐causing missense variants within the genu‐proximal part of calf‐1 (Table ITGB3 mutations so the role of Arg755Gln in the GT phenotype is difficult to predict (cases 35a,b in Nurden et al., Calf‐1 has a β‐structure with nine β‐strands connected to the NH‐1 Table . D'Andre‐1 Table  reported‐1 Table . While A‐1 Table  and S2. Other missense mutations affecting calf‐1 concern residues that locate to β‐strands central to the β‐barrel Figure  but few 4Despite occurring in an a‐priori critical region for changing between resting and activated states, we show that disease‐causing variants within or near the αIIb genu are primarily associated with defects of αIIbβ3 maturation and a loss of surface expression. The genu links the calf‐1 and thigh domains see  of αIIb All 15 GT‐causing missense variants previously identiffied within the amino acid 471‐769 sequence resulted in loss or severely reduced αIIbβ3 expression in platelets Table ; a resulIn silico modeling of the αIIb Asp591Ala mutation in the index case showed a loss of close‐range H‐bonds including Arg551, itself a mutation hotspot for GT being substituted either by Trp or by Gln in several unrelated type I patients Table . Asp591 The disease‐causing effect of αIIbGln626His has a different explanation. Located immediately behind the genu, it is the cause of type I GT in several unrelated families in Europe with a c.1878G>C transversion affecting splicing with loss of exon 28 resulting in truncated mRNA undergoing rapid decay (Jallu et al., ITGA2B and ITGB3 mutation repertoire in the general population (Buitrago et al., A striking feature of the α‐subunit genu is a short, disulfide‐bonded loop first identiffied between residues 746 and 752 in αL and corresponding to residues 633 and 639 in αIIb (see Figure Fundamental to our understanding of the structural effects of genetic variants affecting the αIIb genu is the conformation of the integrin during early αIIbβ3 biosynthesis and much less is known here. Of the 16 disease‐contributing variants that form the basis of our study, all but two give rise to type I GT consistent with a severe block in platelet αIIbβ3 expression; only Ala477Pro and Cys705Arg give rise to type II disease and low amounts of αIIbβ3 on platelets. Crucially, it has been suggested that proαIIbβ3 rapidly adopts a bent conformation during biogenesis (Mitchell, Li, French, & Coller, None of the authors have any conflicts of interest to disclose with respect to the work described in this paper. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Antifreeze proteins (AFPs) enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain’s β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs. Ice binding proteins (IBPs) are characterized by their ability to specifically bind to one or multiple planes of ice crystals . AntifreWhile all AFPs share the similar function of ice binding, their sequences and structures differ widely, making it difficult to infer their molecular detail responsible for this property. The AFPs of Antarctic fish were the first to be discovered  and haveThe ice-binding site (IBS) is the functional region of an AFP. Due to low sequence similarity between AFPs, there are almost no common sequences or structural folds to aid in identifying the IBS. Therefore, the most direct way to determine the IBS of an AFP is using site-directed mutagenesis to systematically study how changes in the property and/or size of residues affects antifreeze activity. Although the overall structures of AFPs vary significantly, it has been found that the IBSs do share common features. IBSs are characterized by a large, relatively flat and hydrophobic plane on the protein surface, and commonly have repeating motifs . It remaFlavobacteriaceae (strain 3519–10) that was isolated from a depth of ~3.5 km in the Vostok Ice Core [Previously we examined the expression, ice binding affinity, and effect on cell viability during freeze-thaw cycling of a bacterial protein , 47, 48. strain 319–10 thaRecombinant IBPv was purified as previously described . The IBPSite-directed mutagenesis was conducted with the Quikchange mutagenesis kit (Agilent Genomics). Primers were designed to overlap with at least 10bp upstream and downstream of the mutation site. The PCR product was treated with DpnI to remove the template and then transformed into DH5α cells. Plasmids were extracted and mutations were confirmed with DNA sequencing. IBPv mutants were purified using similar procedure as wild type.R32 with a = 119.83 Å, c = 367.76 Å.Crystals of the enzyme were obtained using the hanging drop vapor-diffusion method by mixing equal volumes of protein (50 mg/mL concentration in TBS buffer) and the reservoir solution  at 22°C. The crystals grew in approximately one month and belonged to the rhombohedral space group Prior to the data collection, a suitable crystal was dipped for several seconds in a modified mother liquor solution with the addition of 15% glycerol as a cryoprotectant. Diffraction data were collected at 100 K at beamline X6A in the National Synchrotron Light Source of the Brookhaven National Laboratory at a 1.00 Å wavelength. The images were processed and scaled using the HKL2000 program suit . Data coLeIBP from Arctic yeast Leucosporidium sp. for which crystal structure is known [LeIBP per one molecule of IBPv. The molecular replacement procedure was applied to locate a solution using the program MOLREP [LeIBP (PDB accession code 3UYU) was used as a search model. A total of four LeIBP monomers were located in the asymmetric unit of IBP.IBPv shares low sequence identity (29%) with the ice binding protein is known . Accordim MOLREP . A monomThe positioned MR model was refined using the maximum likelihood refinement in REFMAC  with theThe CD spectra of the IBPv, IBPv_a and their mutants were measured from 200nm to 250nm with a Jasco Model J-815 circular dichroism spectrometer. The protein solutions were diluted in TBS to around 0.5mg/mL and placed in a 0.1cm path length cell. Data were collected at 0.5nm bandwidth and the scan speed was 50nm/min. Three scan values were averaged. CD data is available in TH activities of the IBPv, IBPv_a and their mutants were measured with a nanoliter osmometer  connected to a Leica DM LB2 microscope. First, cargille B emersion oil was applied to the sample holes. Then about 20nL of sample was loaded into the oil with a capillary tube connected to a syringe. The temperature was rapidly decreased to below -20°C to initiate freezing process. After ice formation, the temperature was gradually raised until a single ice crystal (diameter in 1–10 μm range) remained in the droplet. The temperature was recorded as the melting temperature. The sample was then slowly cooled until a rapid burst of ice crystal growth was observed. The temperature was recorded as the freezing temperature. The difference between the freezing and melting temperatures was calculated as the TH. TH data is available in LeIBP (PDB: 3UYU) [TisAFP6 (PDB: 3VN3) [TisAFP8 (PDB: 5B5H) [FfIBP (PDB: 4NU2) [ColAFP (PDB: 3WP9) [The structural alignment of IBPv_a, IBPv_b and their homologous proteins, namely B: 3UYU) , TisAFP6B: 3VN3) , TisAFP8B: 5B5H) , FfIBP (B: 4NU2)  and ColAB: 3WP9) , was perB: 3WP9) . The strB: 3WP9) .The 1.75Å IBPv structure was determined by molecular replacement and the overall structure is shown in Ordered water molecules are located around both domains; however, more water molecules are bound to domain B than domain A . PreviouThe IBPv structure suggests that hydrophobic interactions play an important role in protein stability, consistent with our previous observation that IBPv was quite stable at higher temperatures, with a Tm of 53.5°C . As illuEach domain contains one intra-domain disulfide bond, namely C165-C226 in domain A and C244-C443 in domain B . C226 anSeveral studies have shown that IBS of AFPs are flat planes on the protein surfaces . SometimTo confirm the putative IBS, several mutations were generated to introduce a bulky side chain on the IBS  in an attempt to disrupt its flatness and ice binding ability . Our preSince domain B plays the predominant role in TH activity , mutatioIn order to determine the proposed IBS of domain A by mutagenesis, the recombinant domain A (IBPv_a) was used instead of the full length protein, because domain B had much higher TH activity than domain A. Mutating one residue T214 at the proposed IBS and one residue away from the proposed IBS (S201)  into TyrDomain A and B have 44% sequence identities and 61% positive matches, and the crystal structure showed that they both adopt a similar β helical structure . StructuStructural alignment at the IBS of both domains was used to elucidate the structural basis for the differences in ice binding activities. The first row of amino acids on the IBS reside on loops, and are Ala41-Ala212-Ala193-Leu174-Ala147 for domain A, and Ala253-Gly429-Ala410-Ala391-Ala364 for domain B . The secUsing the IBPv_a as a template, residue Leu174 was mutated to Ala. Comparing to the wild type IBPv_a, the mutant IBPv_a L174A significantly increased the TH activity, from 0.65 ± 0.07°C to 0.98 ± 0.08°C at a protein concentration of 0.25mM . This reLeIBP (from fungus Leucosporidium sp. ay30) [TisAFP6 (from fungus Typhula ishikariensis) [TisAFP8 (from fungus Typhula ishikariensis) [FfIBP (from bacterium Flavobacterium frigoris) [ColAFP (from bacterium Colwellia sp. slw05) [LeIBP, TisAFP6, TisAFP8, FfIBP and ColAFP were structurally aligned using MultiSeq [ColAFP [FfIBP [TisAFP8 [To date, several other AFPs that have been determined adopted the similar irregular β helical structure, namely p. ay30) , TisAFP6riensis) , FfIBP (rigoris)  and ColA. slw05) . The strMultiSeq , as showMultiSeq . Blue-wh [ColAFP , FfIBP [P [FfIBP , TisAFP8[TisAFP8  and IBPv[TisAFP8  are cateIn this study, we determined the crystal structure of IBPv, which contains two homologous ice binding domains. This is the first reported crystal structure of a multi-domain AFP. Structural alignment indicates that the domains are structurally similar, with RMSD of 0.68Å on the protein backbone alignment. Each ice binding domain contains an irregular β-helix with a triangular cross-section, and a long α-helix running parallel along one side of the β-helix. A large number of hydrophobic residues were found inside the β-helix and at the interface between the α-helix and β-helix for each domain. One intra-domain disulfide bond was found in each domain as well. These results indicated that hydrophobic interactions and intra-domain disulfide bonds are the major forces that contribute to the overall stability of the protein, which may explain its thermal stability with a denaturation temperature of 53.5°C .AFPs with similar β helical structure were aligned. Despite their high structural similarities, the sequence conservation is relatively low. These β helical AFPs exhibit high sequence conservation on the β helix core, the interface between α helix and β helix, and some turns on the β helix, which all are pivotal in maintaining the overall β helical protein structure. These findings suggested that β helical structure might have its advantageous in supporting the ice binding activity. One major advantage for AFPs to adopt β helical structure is that each coil in the solenoid has an axial rise of about 4.8Å , which pet al recently reported a hydrophilic residue, Ser, on the IBS of TisAFP6 that might partially account for its inferior antifreeze activity compared to its isoform TisAFP8 [AFPs bind specifically, rapidly and irreversibly to ice in aqueous solution , 57. Dif TisAFP8 . Therefo TisAFP8 . More re TisAFP8 –45, 61.  TisAFP8 , 24. In Site-directed mutagenesis confirmed that the proposed ice binding site for each domain is located at the same region on the β-helix for IBPv. Many well aligned water molecules that are hydrogen bonding with the protein backbone were observed on the IBS for each domain. More ordered water molecules were found on the IBS of domain B, consistent with the previous observation that domain B has a much higher antifreeze activity . The resEach domain of IBPv has two rows of water molecules on the IBS that form hydrogen bonds with the protein backbone, and more water molecules are found in the IBS of domain B compared to domain A . The firTwo rows of side chains are well aligned with the two rows of water molecules on the IBS of each domain. The first row of the side chains are located between two rows of water molecules and are mainly small side chains except Leu 174 in domain A. The residues on the second row are mainly hydrophilic residues with hydroxyl groups that may potentially form hydrogen bonds. Close molecular comparison of the two domains revealed that the bulky side chain of Leu 174 on the IBS of domain A may account for its inferior antifreeze activity. The mutant L174A, in which the isobutyl group was replaced by a much smaller methyl group, enhanced the TH of IBPv_a, confirming this hypothesis. In contrast, mutating other small residues on the IBS of domain B into more bulky Leu significantly reduced the TH of IBPv. Therefore, the first row of the small, hydrophobic side chain residues play important roles in allowing two rows of water molecules to form hydrogen bonding interactions with the protein backbone. These findings suggest that the non-hindering effect at the IBS is vital for the ice binding of AFPs.Although the L174A mutation produced a TH for domain A that was comparable to domain B, it did not increase, and even slightly impaired, the TH of the full length IBPv. Our previous work showed that domain B contributed the majority portion of the TH of IBPv . Thus a S1 FigS201 in Domain A is distal to the predicted IBS.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table3 deg cm2 · dmol-1.Mean residue molar ellipticity are displayed in 10(DOCX)Click here for additional data file.


A domain called the 'Conserved region in the middle' is responsible for target recognition in the TORC2 complex in fission yeast and the mTORC2 complex in mammals. Related research article Tatebe H, Murayama S, Yonekura T, Hatano T, Richter D, Furuya T, Kataoka S, Furuita K, Kojima C, Shiozaki K. 2017. Substrate specificity of TOR complex 2 is determined by a ubiquitin-fold domain of the Sin1 subunit. eLife6:e19594. doi: 10.7554/eLife.19594Cellular growth is a tightly regulated process that depends on the availability of energy and nutrients. Moreover, in multicellular species, the proliferation of cells must be coordinated across tissues and organs. Protein kinases called target of rapamycin (TOR) proteins and their mammalian ortholog mTOR have a central role in growth regulation. These proteins exert their function in two complexes called TORC1 and TORC2 . These complexes act as regulatory hubs that integrate input signals concerning the availability of energy and nutrients or the presence of growth factors. With their outputs, the mTOR complexes control metabolism and protein biosynthesis, and influence cell cycle progression, autophagy and cytoskeletal organization .TOR and mTOR are very large proteins (containing approximately 2300–2600 amino acids) and are members of the PIKK family of regulatory kinases. Both mTOR complexes comprise mTOR and a protein called mLST8 . mTORC1 In mTORC1, the Raptor N-terminal conserved (RNC) domain is known to be involved in the binding of linear recognition motifs in target proteins . Recent However, less is known about target recognition in TORC2 and mTORC2. Now, in eLife, researchers at the Nara Institute of Science and Technology, Osaka University and the University of California, Davis -- including Hisashi Tatebe and Shinichi Murayama as joint first authors, and Tatebe and Kazuhiro Shiozaki as corresponding authors -- report the results of a series of studies on human SIN1 and its fission yeast ortholog Sin1, that shed light on the conserved role of these proteins in target binding .SIN1 and its orthologs are known to contain four domains : a variaBased on earlier studies , SIN1 maTatebe et al. also report an NMR structure for the CRIM domain in fission yeast that reveals a ubiquitin-like fold with a prominent acidic loop. Mutational analysis confirms that target binding depends on the integrity of the CRIM domain and strongly suggests that the acidic loop is involved in binding.With the functional role and structure of the CRIM domain now firmly established, researchers can start to address the many remaining questions about TORC2 target recognition. The NMR structure of the CRIM domain provides an excellent platform for a detailed analysis of the recognition of specific motifs in target substrates by SIN1. However, studies of the overall integration of SIN1 into mTORC2 will be required to address the consequences of SIN1-target binding for subsequent phosphorylation. Finally, work on target recognition in mTOR complexes together with recent structural and functional studies on other PIKK family kinases  may reve


R2 = 0.97, PRESS = 2.14; R2 = 0.96, PRESS = 0.69; R2 = 0.95, PRESS = 1.27; R2 = 0.94, PRESS = 0.76, for content of proteins, lipids, ash, and moisture level, respectively. Best results of CSR models were obtained for protein, ash, and crude fat .Samples of wheat, spelt, rye, and triticale flours produced by different Polish mills were studied by both classic chemical methods and FT-IR MIR spectroscopy. An attempt was made to statistically correlate FT-IR spectral data with reference data with regard to content of various components, for example, proteins, fats, ash, and fatty acids as well as properties such as moisture, falling number, and energetic value. This correlation resulted in calibrated and validated statistical models for versatile evaluation of unknown flour samples. The calibration data set was used to construct calibration models with use of the CSR and the PLS with the leave one-out, cross-validation techniques. The calibrated models were validated with a validation data set. The results obtained confirmed that application of statistical models based on MIR spectral data is a robust, accurate, precise, rapid, inexpensive, and convenient methodology for determination of flour characteristics, as well as for detection of content of selected flour ingredients. The obtained models' characteristics were as follows:  Flour, a product obtained in the process of grain milling, is one of the major raw materials in food industry. Flour is indispensable for production of a wide variety of staple products such as bread, pasta, cakes, and biscuits. Flour must be of satisfactory quality for processing to obtain acceptable end products.Basic technological parameters of flour are content and quality of proteins, content of ash, amylolytic activity expressed as falling number or moisture. Lipids, another flour component, is also important, especially due to calorific value of flour. Gluten protein in flour creates a three-dimensional structure during dough mixing. Flour therefore must contain adequate amount of quality protein, which will hold bloated starch granules and gas bubbles within the dough structure. Bread and yeast cakes demand flour of high protein content, while other cake products can be made with flour of low protein content. Ash content does not really influence usable properties of flour; it is, however, the basic parameter for flour classification. Moisture values inform us indirectly about flour storage conditions. Excessive moisture facilitates growing of moulds; inadequate moisture, under 12%, promotes rancidity in fats present in flour , 0.0014 is content of nitrogen [g] corresponding to 1 mL of 0.1 mol L−1 HCl solution, 200 is dilution factor.Protein content was determined indirectly by measuring the total nitrogen content. A flour sample (0.5 g) was mineralized for 30–60 minutes with 12 mL of 35% sulphuric acid (VI) and catalyst  to extract lipids with petroleum ether. Extraction was performed during 120 minutes at 155°C and proceeded in four steps: boiling, rinsing, solvent recovery, and drying. Crude fat content was calculated as follows :(2)m2−m1Ash content was determined by weighting remnants of the burning process of a 5.0 g flour sample conducted at 900°C for 1 hour in muffle furnace  .Falling number was measured according to the Hagberg-Perten method  by measuMoisture was measured by the weighting method according to the Polish standard . A 10 g K is calorimetric constant [J/°C], T2, T3 are temperatures of heating balance [°C], k is correction for the calorimeter-environment heat exchange, m is sample mass [g].Calorific value was measured with a KL-12Mn calorimeter (Precyzja Bit Company). A flour sample pellet of an exact weight was placed in a fixed-volume bomb and burnt in a pressurized environment high in pure oxygen. Based on the temperature increase, the sample mass, and the bomb constant, the heat of combustion was calculated as follows:k was calculated as follows:n is time of the main period in minutes (time of the increase of temperature from T2 to T3), T1, T4 are characteristic temperatures of heating balance.μm. The column temperature was programmed to increase from 60 to 230°C and the injector and detector ports were set at 225°C and 250°C, respectively. Detailed procedure is presented in Reder et al. [Fatty acids content was determined by gas chromatography with a Shimadzu model GC-17A gas chromatograph equipped with a flame-ionization detector and a 30-metre capillary column of 0.22 mm, that is, with a film thickness of 0.25 r et al. .−1. KBr matrix pellets were prepared by mixing 300 mg of KBr with 1 mg of sample in laboratory ball mill. Then mixture was pressed in laboratory press with press 10 tones. Ready pellet was placed in measuring holder-dedicated accessory of System 2000 spectrometer and placed in measuring chamber. Average spectrum was considered final. The resolution was 4 cm−1 and the shift velocity 2 cm s−1. DTGS (deuterated triglycine sulphate) detector is a part of used spectrometer.The 2000 System Perkin Elmer instrument operated by PEGRAMS software running on Windows 95 platform was used to register FT-IR spectra. The transmission technique was applied to conduct 25 scans for each of the studied flours in the spectral range of 4000–370 cmStatistical procedures were carried out using Statgraphics Plus 5.1 software. Statistically significant differences between flours were calculated with the one-way ANOVA method (Tukey's procedure). Spectral data, for example, integral intensity of 12 selected bands, were correlated with the content of the selected flour ingredient.TQ Analyst running on Windows XP platform was used to search for the best statistical models correlating the spectral and the chemical data. The cross-validation diagnostics with one-left-out procedure was used to validate the models. The spectra were automatically normalized and mean-centered. Bond frequencies and intensities were used to calibrate statistical models. Maximal number of 20 factors was set for the tested models. For all parameters identical spectral pretreatment was applied.In Poland, chemical and physical composition of flours is regulated by the two Polish standards , 37 for p < 0.05 at confidence level 95%) in protein content were observed for all groups analyzed flours.Among the studied flours, the highest content of protein was observed for spelt flour. Its value increased with the flour type and ranged from 12.07 to 12.61%. The lowest protein content was in rye flour. This is obviously related to lower protein content in the rye grain than in the wheat and spelt grains. Protein content of 13.39% in bran was higher than those detected in flours. Statistically significant differences  and higher types  of flours were statistically significant. The higher flour type the higher content of crude fat. This is related to distribution of lipids in the grain. The higher content of lipid is characteristic for the germ and the aleurone layer. As low type flour is made of material containing endosperm, the lipid level is at its lowest. On the other hand, bran which is made of mostly of the external part of the grain contains more lipids.According to the standard , ash is The studied flours differed in ash content. The high content of ash was characteristic for bran while wheat flours of lowest types, 550 and 650, characterize the lowest content of this parameter. Overall, the level of ash was the highest in spelt flour which suggests the highest content of mineral elements in the spelt grain compared to the wheat, rye, and triticale grain. Differences between spelt, wheat, rye, and triticale flour type 2000 were statistically significant. Wheat flour type 2000 eco originated from ecological source had statistically significant higher content of ash as compared to the wheat flour same type originated from the nonecological source.The falling number values varied between 107 and 317 seconds. The highest value was observed for spelt flour, while the lowest for rye flour. Optimal falling number was observed for wheat flours: 243 and 248 for 2000 type and 242 and 261 for 550 and 650 types, respectively. The highest amylolitical activity was observed in the cases of rye and triticale flours which confirmed previous literature data: Makowska and Stachowiak  and SłowMoisture percentages of the analyzed flours varied between 11.1 and 13.7%. The highest value was observed for wheat flour type 550. The Polish standard allows for the moisture percentage below 15%. In the case of bran, moisture level should not exceed 11% .The method of determination of calorific value relies on temperature measurement during the isochoric transformation. The deciding components for the calorific value of the studied flours were ordered as follows: lipids, proteins, and carbohydrates with energy equivalents of 39.55 J/g; 26.65 J/g; and 17.37 J/g, respectively. Therefore, flours with the highest content of lipids and proteins should have the highest calorific value which was confirmed by the measurements. The spelt products had the maximum calorific value. For wheat, rye, and triticale flours an increase in combustion value was observed along with the increase in the type of flour. The calorific values of the studied flours were not poles apart, although some marked differences were registered. The lowest value (15477 J/g) was obtained for triticale flour type 700, while the highest (16778 J/g) for spelt flour type 1100.The experimental determination of protein, fat, ash, and fatty acids contents as well as properties such as moisture, the falling number, and energetic value in flour is laborious and time-consuming. The literature contains a lot of examples of correlation of various characteristics with IR spectra in different foodstuffs –43. High−1 is generated by stretching vibration of O–H bond. Bands in the range 3000–2800 cm−1 are assigned to stretching vibrations of C–H bond. Spectral region between 1500 and 900 cm−1 is called fingerprint region because of the unique patterns characteristic for given sample. The assignment of spectral bands to vibrations generating these bands is presented on The spectral data were correlated with the reference results in two different ways. The classical square regression (CSR) and partially least square (PLS) techniques were applied. The spectral ranges used for those analyses are presented in R2) were used as a statistical measure of the model fitting.The linear regression analysis presents the relation between two variables: the ratio of two selected spectral bands (an dependent variable) and the content of a given chemical (independent variable). Coefficients of determination  may have wide practical applications, at the very least in the preliminary assessment of technological value of flour. Remaining flour parameters, listed in chapter, were also tested to find robust models correlating spectral data with given feature. Unfortunately, statistically significant models were not obtained.The samples in the calibration/validation set were of slightly different chemical compositions due to different grain sources and milling processes. Those differences were both quantitative and qualitative in nature because the spectral variations observed manifested them as increase/decrease in absorbance in a given spectral range .R2) between the actual and predicted values , the Root Mean Square Error of Calibration (RMSEC), the Root Mean Square Error of Prediction (RMSEP), and the Prediction Residual Error Sum of Squares (PRESS) values. The higher R2 and the lower RMSEC, RMSEP, and PRESS, the more precise the model. Those statistical parameters have been used previously for different foodstuffs analyses [Optimal calibration equations were explored using the PLS-1 regression. Optimal number of factors in constructing a model is very important since adding too many (more factors) which might come from noise in the data  may decrease prediction strength of a model. On the other hand, if too few factors are used , prediction accuracy for unknown samples will suffer as not enough factors are used to express all spectral variations influencing a given value. Therefore, it is very important to define a model that consists of the right number of factors to model a given value properly . The optanalyses , 44, 45.R2 values with a given number of factors numbered I, II, III, and IV were calculated for proteins, lipids, ash, and moisture level, respectively. The calculated models' parameters were as follows: R2 = 0.97, PRESS = 2.14, RMSEC = 0.22, RMSEP = 0.32, and factors number = 10; R2 = 0.96, PRESS = 0.69, RMSEC = 0.11, RMSEP = 0.13, and factors number = 3; R2 = 0.95, PRESS = 1.27, RMSEC = 0.38, RMSEP = 0.55, and factors number = 2; and R2 = 0.94, PRESS = 0.76, RMSEC = 0.11, RMSEP = 0.15, and factors number = 5, for models I, II, III, and IV, respectively. Statistical parameters calculated for every model are presented in The models of minimal RMPSEC, RMSEP, and PRESS and maximal −1; 1834–1583 cm−1; 1583–1494 cm−1 and 1494–1280 cm−1. Within the regions selected, there were 497 spectral data points.Spectral ranges for all models were assigned based on initial visual inspection which resulted in detection of most distinct differences in spectra. For model I the following spectral regions were selected: 3025–2800 cm−1; 1834–1583 cm−1; 1279–1221 cm−1; and 952–886 cm−1. Within the regions selected, there were 1194 spectral data points.For model II the following spectral regions were selected: 3846–3027 cm−1; 1197–952 cm−1; 952–886 cm−1. Within the regions selected, there were 369 spectral data points.For model III the following spectral regions were selected: 1279–1221 cm−1 and 1279–1221 cm−1. Within the regions selected, there were 877 spectral data points.For model IV the following spectral regions were selected: 3846–3027 cmTo construct, validate, and test each model, the total number of 60 samples (5 repetitions for each of 11 flour types and 1 bran) was randomly divided into two groups of 48 and 12 elements, separately for each measured parameter. Each of the 48-element group was used for model calibration and validation. The 12-element groups, containing precisely one sample of each studied flour type and bran, were treated as unknown independent samples not included in the model calibration and used to test the obtained models. It meant that those flour samples could be any samples purchased in any shop at any time.The values of the studied parameters  of these samples were predicted with the created model while their actual values were measured by standard methods. Subsequently, the two sets of values were correlated. There was a linear correlation between the actual and predicted values of the studied parameters .R2 = 0.97 and slope, S = 0.93. The parameters of the linear correlation between the model-predicted and actual values are as follows: R2 = 0.96, S = 0.91; R2 = 0.95, S = 0.93; R2 = 0.94, S = 0.91 for lipids, ash, and moisture, respectively.(1) CSR procedure produced statistically significant relationship for selected bands intensities ratio and level of protein, lipids, ash, linolenic acid, and falling number.(2) Four statistically significant models calibrated with use of PLS were obtained for content of proteins, lipids, and ash well as moisture level. Those models can serve to studied flours. Models parameters are as follows:R2 = 0.97, PRESS = 2.14, and factors number = 10.Model I (protein content): R2 = 0.96, PRESS = 0.69, and factors number = 3.Model II (lipids content): R2 = 0.95, PRESS = 1.27, and factors number = 2.Model III (ash content): R2 = 0.94, PRESS = 0.76, and factors number = 5.Model IV (moisture level): (3) Interestingly, in the case of selected parameters, for example, linolenic acid, the simpler CSR procedure produced better results than the more sophisticated PLS technique.(4) In the case of calorific value no statistically significant correlations with spectral data were determined.(5) Compering the results obtained by meaning of two different statistical techniques one can conclude (see Tables


Here we show that mutations in the transmembrane domain of TRPV6 can result in conversion from a domain-swapped to non-swapped fold. These results reveal structural determinants of domain swapping and raise the possibility that a single ion channel subtype can fold into either arrangement  Structurally, TRP channels are assemblies of four identical subunits arranged symmetrically around the pore axis6. The transmembrane domain of TRP channels includes six transmembrane helices (S1–S6) and a pore loop (P-loop) between S5 and S6. The first four helices (S1–S4 domain) form a bundle that plays the role of voltage sensing domain in voltage-gated potassium channels7. S5, P-loop and S6 form the KcsA channel-like pore domain, with the ion conduction pathway lined by the S6 helices and extracellular loops connecting the P-loop helix to S5 and S6. In the cryo-EM reconstructions of the vanilloid subtype TRP channels TRPV12 and TRPV26, the S1–S4 domain of one subunit is packed against the pore domain of the neighboring subunit, producing a domain-swapped arrangement similar to the voltage-gated potassium channel chimera Kv1.2–2.18.Transient receptor potential (TRP) channels comprise a large and functionally versatile superfamily of cation permeable ion channels that respond to a myriad of sensory modalities2+-selective and play an important role in calcium homeostasis10. Altered expression of TRPV6 is associated with numerous human diseases, including various forms of cancer11. Accordingly, TRPV6 has emerged as an important target for drug design12. We recently solved the structure of a TRPV6 construct engineered to produce well diffracting crystals5. On the basis of electron density and cysteine crosslinking experiments, we built a model of TRPV6 in which the S1–S4 domain of one subunit is packed against the pore domain of the same subunit, creating a non-swapped architecture distinct from TRPV12 and TRPV26. Subsequent studies of HCN13, CNG14, Slo115, Slo2.216 and Eag117 have revealed the prevalence of non-swapped transmembrane domain architecture in tetrameric ion channels.TRPV6 and its close relative TRPV5 are TRP channels that are distinctly Ca2 and TRPV26, and relatively high sequence conservation in the transmembrane region  affected the domain arrangement. Compared to wild type, TRPV6cryst has a 59-residue C-terminal truncation and four point mutations, including L495Q, which resides in the S5 transmembrane helix  and S6-TRP helix (residues 577–589) gave a root mean square deviation (RMSD) of 0.865 Å.To crystallize TRPV6*, we used the same expression system, purification protocol and crystallization conditions as for TRPV6st Table  and the st Table . Despitecryst and confirmed its non-swapped conformation. Notably, we were able to collect new crystallographic data, where both the S4–S5 and S6-TRP helix connectivity in TRPV6cryst is unambiguously resolved in the 2Fo-Fc and Fo-Fc omit maps  was missing. Similarly, both the 2Fo-Fc and robust Fo-Fc omit maps for TRPV6* demonstrated completely different connectivity of transmembrane domains  followed by eGFP and streptavidin affinity tag (WSHPQFEK), for the expression in suspension of baculovirus-transduced HEK293 GnTI– cells. 48–72 hours after transduction with P2 virus, cells were harvested, washed with PBS at pH 8.0 and after sonication, cellular membranes were prepared. The protein was extracted from cellular membranes using 20 mM n-dodecyl-β-D-maltopyranoside, and purified using Strep-Tactin affinity chromatography followed by size exclusion chromatography.TRPV6 constructs described in this paper were expressed and purified as previously described for the TRPV6cryst5, which included a reservoir solution consisting of 20–24% PEG 350 MME, 50 mM NaCl and 50 mM Tris-HCl pH 8.0–8.5 in hanging drop configuration at 20 °C. Prior to crystallization using hanging drop method, the protein was subjected to ultracentrifugation  to remove the aggregated protein. Crystals were cryoprotected by serial transfer into the buffer composed of 100 mM NaCl, 100 mM Tris-HCl pH 8.2, 0.5 mM DDM and 50 mM ammonium formate, containing increasing concentration of PEG 350 MME, with maximum concentration of up to 33–36%, and then flash frozen in liquid nitrogen. To obtain crystals with Ca2+ or Gd3+, protein was incubated with 10 mM CaCl2 or 1 mM GdCl3, respectively, for at least 1 hour at 4 °C prior to crystallization.Optimized crystals of purified TRPV6* or TRPV6*-del1 were grown in the same condition as crystals of TRPV618 or HKL200019. The initial phase information and structures were solved by molecular replacement using Phaser20 and the structure of rat TRPV6cryst5 (PDB ID: 5IWK) as a search probe. Most of TRPV6*, including the ankyrin domain, S1–S4, pore module and C-terminal hook, was nearly identical to the TRPV6cryst; the rest of the structure was built using the omit map as a guide. The robust electron density for the S4–S5 linker was evident from the initial phases obtained by molecular replacement, and map features improved further during refinement. The model was refined by alternating cycles of building in COOT21 and automatic refinement in Phenix22 and Refmac23. All structural figures were made using the PyMOL Molecular Graphics System .X-ray diffraction data collected at APS (beamlines 24-ID-C/E), NSLSII (beamline 17-ID) or ALS (beamline 5.0.2) were indexed, integrated and scaled using XDS2+ measurements from HEK cells expressing rat TRPV6 or TRPV6cryst or TRPV6* were performed as described previously5. All Fura2-AM-based fluorescence measurements were conducted using spectrofluorometer QuantaMasterTM 40  at room temperature in a quartz cuvette under constant stirring.The intracellular Ca


Molecular architecture of the 90S small subunit pre-ribosome. Published 28, February 2017In the published article, two typos have been corrected.In the second sentence of the third paragraph of the chapter "UTPA", the text "The crystal structures of the N-terminal region (N domain) of Utp10 in complex with a C-terminal peptide of Utp17 and a middle region (M domain) of Utp10 were docked into two separated places in the density " has been replaced with "The crystal structures of the N-terminal region (N domain) of Utp10 in complex with a C-terminal peptide of Utp17 and a middle region (M domain) of Utp10 were docked into two separated places in the density ".In the second sentence of the fourth paragraph of the chapter "The central domain", the text "The density, although weak, was well fitted by the crystal structure of the C-terminal tetratricopeptide repeat (TPR) domain of Rrp5 " has been replaced with "The density, although weak, was well fitted by the crystal structure of the C-terminal tetratricopeptide repeat (TPR) domain of Rrp5 ".The article has been corrected accordingly.


Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2’s FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of  Target of rapamycin (TOR) kinase operates within two distinct multiprotein complexes named TORC1 and TORC2. Here the authors report a cryo-EM structure of TORC2, establish its subunit organization, providing a rationale for TORC2’s rapamycin insensitivity and the mutually exclusive inclusion of Avo3/Rictor or Raptor within their respective TOR complex. TOR) genes were first discovered in an elegant genetic dissection of Saccharomyces cerevisiae mutants exhibiting resistance to rapamycin1, a macrolide produced by a soil bacterium from Easter Island2. This study also demonstrated that rapamycin requires a co-factor for toxicity, a 12 kDa proline isomerase named FKBP12 for FK506 (a macrolide similar to rapamycin)-binding protein. Subsequent to its discovery in yeast, a mammalian ortholog (mTOR) was described5 and indeed it is now appreciated that TOR is widely conserved in eukaryotes6. Unlike yeast, which encodes two TOR genes, TOR1 and TOR2, higher eukaryotes encode only a single TOR gene. The amino acid sequence of Tor initially suggested that it would function as a lipid kinase8. However, Tor is a Ser/Thr protein kinase and the founding member of a family of related proteins including DNA-PKcs, SMG1, ATR and ATM, known as phosphatidyl-inositol-kinase-like kinases (PIKKs), which all resemble phosphatidyl-inositol kinases but are in fact Ser/Thr protein kinases9. Among the PIKKs, only Tor, at its FKBP12-rapamycin-binding domain (FRB) in the N-terminus of the kinase domain, can be bound, and thus inhibited, by a complex of rapamycin and FKBP12.The target of rapamycin  is composed of either Tor1 or Tor2 (mTOR), Kog1 (Raptor), Lst8 (mLst8/GβL) and Tco89 (no ortholog). Rapamycin-FKBP12 binding to the FRB of Tor in TORC1 inhibits kinase activity via steric occlusion of the active site19. In humans, abnormal mTORC1 activity is associated with a wide range of clinical presentations for which rapamycin or related compounds are often indicated20. These include cancer, loss of metabolic homoeostasis, immune dysfunction and ageing. Rapamycin has also been a useful tool for basic scientists to study TORC1 signalling. Indeed, the mechanisms by which upstream regulators  regulate TORC1, as well as the downstream targets by which TORC1 regulates its distal effectors  are becoming increasingly well understood23.Biochemical purification of Tor1 and Tor2 from yeast demonstrated that Tor exists in two different multiprotein complexes named TORC1 and TORC224. Like mTORC1, mTORC2 dysregulation is implicated in cancer and metabolic perturbations25, but unlike mTORC1, specific small-molecule inhibitors of mTORC2 are not presently available. Consequently, the mechanisms of upstream regulators  and downstream effectors  of TORC2 are relatively less well characterized30. Further insights into the TOR complexes have come from recent structural studies that have revealed the molecular architecture of TORC1. From N-terminus to C-terminus, Tor contains two blocks of HEAT  repeats, a block of TPR (tetratricopeptide) repeats known as the FAT  domain, and the kinase domain, which includes the FRB domain inserted in its N-terminal part and the FAT C-terminal (FATC) domain31  is composed of Tor2 (mTOR), Lst8 (mLst8/GβL), Avo1 (hSin1), Avo2 (no ortholog), Avo3 (Rictor) and Bit61 or its paralog Bit2 (Protor1 or Protor2). The FRB domain of Tor2 has been suggested to be inaccessible in TORC2, which would explain why the kinase activity of this complex is insensitive to acute treatment with rapamycinn31 Fig. . Lst8 isn31 Fig. . A cryst34. These structures revealed that the N-terminal HEAT blocks assemble into two α-helical solenoid super-structures, respectively, referred to as the “spiral”33 or “horn”32 and the “bridge” that is followed by an extended linker referred to as the “railing”  and crystallographic studies, using proteins from mammals and thermo-tolerant yeast, have yielded additional insight into the organization of TORC1ng” Fig. . These sS. cerevisiae TORC2 using negative stain EM and crosslinking/mass spectrometry (XL/MS)24. This work defined a rhombohedral shape for TORC2, globally similar to, but slightly larger than mTORC135. We found that deletion of the C-terminal 157 amino acids of Avo3 made the kinase activity of TORC2 sensitive to inhibition with rapamycin-FKBP12 and accordingly, the C-terminus of Avo3 was suggested to lie in proximity to the FRB domain of Tor228. The PH domain located in the C-terminus of Avo1 was placed at the obtuse edge of the rhombohedron. Curiously, in the recent high-resolution mTORC1 structures, this region is clearly occupied by mLst834.We provided preliminary insights into the molecular architecture of S. cerevisiae TORC2 at 7.9 Å resolution. TORC2 adopts a twofold symmetric rhombohedral structure comprising two copies of each subunit. We reveal the subunit architecture of the complex, the topology of Lst8 and Tor2 in the complex and the interactions at the dimer interface. We find that Avo3 interacts with Tor2 in a manner analogous to the interaction of Raptor with mTOR in mTORC1, sterically excluding Kog1/Raptor incorporation in TORC2. Avo3 additionally occupies volume adjacent to the Tor2 FRB domain that prevents rapamycin-FKBP12 binding and thus renders TORC2 rapamycin insensitive and serves to further restrict access to the already recessed active site. Density that we attribute to Avo1 resolves poorly, suggesting that this subunit is highly mobile. In agreement with XL/MS experiments24, we propose that the PH domain of Avo1 appears to float above Lst8 and that its conserved-region-in-the-middle (CRIM) domain lines the active site pocket. We corroborate this localization by subunit localization experiments using negative stain EM. Our placement of the CRIM domain is consistent with its proposed role in substrate recruitment36.Here, we describe the cryo-EM structure of S. cerevisiae cell extract via protein-A affinity purification of Bit61-TAP from a Bit2 knockout strain  Tor-Lst8 dimer33   in TORC2 is very similar to the Tor conformation in the Km Tor-Lst8 dimer structure33,  corresponding to part of the spiral domain and residues in the bridge domain  structure of the 16 kDa CRIM domain of the Schizosaccharomyces pombe Avo1 ortholog, Sin138, high-resolution structures of TORC2-specific subunits are lacking.Beyond crystal structures of the 13 kDa pleckstrin homology (PH) domains of 24. These suggested that: Avo1 is located in the Thumb, Avo3 occupies the central part of the complex including regions close to the FRB domain; Bit61 forms the acute angle of the rhombohedron, which corresponds to Finger2 in the negative stain EM reconstruction . We built a homology model for Avo224. Accordingly, in the cryo-EM structure, we attribute the density in the central part of the complex, next to the Tor2 proteins, to Avo3. We built a partial Avo3 model fitting the well-resolved tube-shaped helices in the map  in the same protomer and the spiral of Tor2 (helices Nα27 and Nα29) in the other protomer  and Lst8  and 1 mM PMSF). After centrifugation, TORC2 was purified from the supernatant by ProteinA affinity purification using IgG-coated dynabeads . After washing , TORC2 was released from the beads by incubation with TEV protease (1 h at 18 °C).TORC2 was prepared through pull-down of TAP-tagged Bit61 from yeast extracts lacking Bit2Holey carbon grids  were coated with a thin continuous carbon foil on top. Glow-discharged grids were incubated onto drops of 10–15 μl of TORC2 at 4 °C in order to increase the particle concentration on the grid. After 10 min incubation, the grids were blotted for 2–3 s at 4 °C and 100% relative humidity inside a Vitrobot Mark IV chamber  and subsequently plunge-frozen in liquid ethane.2 were recorded at a nominal magnification of ×59,000 corresponding to a pixel size of 1.38 Å and a total exposure time of 2.3 s. Images were acquired with a defocus range of −2 μm to −4 μm in steps of 0.1 μm. A second data set was collected from the same microscope using a K2 Summit detector (Gatan) with an energy filter. A total of 2847 dose-fractionated movies each containing 40 frames (0.5 s per frame) with a accumulated total dose of 47 e/Å2 were recorded semi-automatically using SerialEM44 at a nominal magnification of ×105,000 in super-resolution mode. Images were recorded with a defocus range of −1.5 μm to −3.5 μm. Subsequently, cropping in Fourier space resulted in a pixel size of 1.35 Å.Single-particle cryo-EM data were collected using a FEI TITAN Krios microscope operated at 300 kV at EMBL Heidelberg. The data were recorded on a FEI Falcon II DED using automated data acquisition software (FEI EPU). A total of 4189 dose-fractionated movies each containing 40 frames with an accumulated total dose of 50 e/Å45. The individual movie frames from the K2 Summit detector were gain-corrected, aligned with the “patch” option, dose-weighted and summed using the MotionCor2 program46. The CTF parameters for both data sets were estimated from CTFFIND447, integrated in Relion1.448.The movie frames from the Falcon II DED were aligned, dose-filtered and summed in Unblur1.0.249 with Gauss option with the box size of 320 pixels. All automatically picked particles were subjected to two rounds of 2D classification in Relion resulting in a pool of 111,196 good quality particles for data set 1 . The TORC2 negative stain reconstruction  was filtered to 60 Å and used as initial reference for 3D classification. At every step of classification, the data were grouped into two volumes  = 0.143 criterion50  and the 16-kDa CRIM domain . SwissModel55 was used to build a homology model for Avo1_CRIM based on the NMR structure of the Sin1 CRIM domain from fission yeast . The Avo1_CRIM model (residues 647–792) fitted into the density between the Tor2 FRB domain and Lst8 . The N-terminal part of Avo2 has been predicted to contains five ankyrin repeats (residues 4–171)39. The homology model of the N-terminal part of Avo2 (aa 4–163) was generated in Phyre2 using the pdb structure 1n11 (chain A) as template. The Avo2 model fits well into the density previously assigned to Avo224 connecting the Tor2 spiral domain on one side and the Tor2 C-terminal domain (cc of 0.94). For Avo3, a set of 17 helices of average length of 20 amino acids could be modelled into EM density contoured at 7.5 sigma. Secondary structure prediction suggests that the middle part of Avo3 contains an armadillo-like helical domain. Accordingly, we assign these alpha helices to the Avo3 armadillo-like domain. Moreover, the localization of these helices agrees with the predicted central position of Avo3 deduced from XL-MS and from negative stain EM domain localization experiments24. The partial model of TORC2 was refined against the density in real_space using the “phenix.real_space_refine” program57. The cross-correlation values for the docked atomic coordinates into the corresponding subunit EM map are listed in Supplementary Table 58  .The atomic model derived from the 6.1 Å map of the The cryo-EM map is available from the Electron Microscopy Data Band with accession code EMD-3896. The partial atomic model has been deposited in the Protein Data Bank with the accession code 6EMK. The data that support the findings of this study are available from the corresponding author upon request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


The 004 peak (c-domain) position shifts to a lower 2θ angle, which demonstrates the elongation of the c-axis lattice parameter of the c-domain under an applied electric field. In contrast, the 400 peak (a-domain) shifts in the opposite direction (higher angle), thus indicating a decrease in the a-axis lattice parameter of the a-domain. 90° domain switching from (100) to (001) orientations (from a-domain to c-domain) was observed by a change in the intensities of the 400 and 004 diffraction peaks by applying a high-speed pulsed electric field 200 ns in width. This change also accompanied a tilt in the angles of each domain from the substrate surface normal direction. This behaviour proved that the 90° domain switched within 40 ns under a high-speed pulsed electric field. Direct observation of such high-speed switching opens the way to design piezo-MEMS devices for high-frequency operation.Ferroelastic domain switching significantly affects piezoelectric properties in ferroelectric materials. The ferroelastic domain switching and the lattice deformation of both  It has been pointed out that the electromechanical response of Pb(Zrx1−Tix)O3 is composed of not only lattice elongation  but also extrinsic contributions, which include the motion of the domain walls, separating ferroelastic domains (non-180° domain motion) under an applied electric field, for example, switching from (100)- to (001)-orientated domains (90° domain switching) in tetragonal Pb(Zrx1−Tix)O3 films2. In particular, the extrinsic contributions are responsible for more than 50% of the total piezoelectric response, as well as the dielectric response3. Therefore, non-180° ferroelastic domain switching induced by the application of an electric field is a significant feature and is required for the design of high-performance piezoelectric devises for MEMS applications. Much efforts have been devoted to clarify the dynamics of the non-180° ferroelastic domain in epitaxial Pb(Zrx1−Tix)O3 films9. The change in the non-180° ferroelastic domain structure with the application of an electric field has been investigated using various techniques, such as transmission electron microscopy (TEM)9, piezoresponse force microscopy (PFM)8, and X-ray diffraction (XRD)5 for polydomain (100)/(001)-oriented epitaxial tetragonal Pb(Zrx1−Tix)O3 films. The domain structures strongly depend on the film thickness and substrate12, and the Zr/(Zr + Ti) ratio in the films13, as well as the poling treatment.Ferroelectric materials have been applied to many electronic devices, including sensors and actuators. Because of their superior piezoelectric and ferroelectric properties, Pb(Zrx1−Tix)O3 films have been measured by in-situ XRD measurement17. Lattice elongation and 180° domain wall motion were observed at sub-nanosecond timescales for (001)-oriented Pb(Zrx1−Tix)O3 thin films15, and it has been reported that non-180° ferroelastic domains can be switched by application of an electric field on the order of hundreds of nanoseconds17 in ferroelectric films. However, it has not been clarified yet how fast does the 90° domain switching proceed - in particular, the relationship between the lattice elongation and geometrical domain structure deformation in tetragonal Pb(Zrx1−Tix)O3 thin films has not been determined from a response speed point of view.The dynamics of the crystal lattice while applying an electric field in Pb(Zr0.4Ti0.6)O3 films, which were subjected to an applied rectangular electric field pulses of 200-ns and rest time of 800 ns. The average lattice parameters and domain structure of the films under this electric field was then determined using a time-resolved in-situ synchrotron X-ray diffraction system in conjunction with a high-speed pulse generator. We show that in contrast to conventionally accepted scenario of sluggish nature of ferroelastic domain switching as pointed out in ceramics18, simultaneous lattice deformation (c-axis and a-axis) and ferroelastic domain switching  occurs on the order of sub-microseconds.In the present study, we investigated the change in lattice parameters and domain structure for 600-nm thick (100)/(001)-oriented epitaxial tetragonal Pb(Zrθ- ω mappings around the PZT 400 and 004 diffraction peaks of (100)/(001)-oriented epitaxial PZT films grown on (100)cSrRuO3//(100)KTaO3 substrates are shown in Fig. 400 diffractions as twin peaks (a-domains) are observed in Fig. 3400c and KTaO3400 diffraction peaks, while split PZT 004 diffractions as twin peaks (c-domains) are observed in Fig. 24. The twin peaks split in the rocking curve direction (ω- axis), the horizontal axis in Fig. 12. The tilting angles of c-domains (β angle) and a-domains (α angle) can be interpreted by considering that the films are required to be attached to a flat surface substrate, as illustrated in Fig. α = 0.41° and β = 1.96° were obtained from the 2θ- ω mappings shown in Fig. c-domain (Vc)22 and the tetragonality (c/a ratio), as described in supplementary materials12.High-resolution X-ray diffraction (HRXRD) 2θ-ω scans for PZT 004  films, 600 nm in thickness, grown on (100)cSrRuO3-coated (100) KTaO3 substrates by pulsed-metal organic chemical vapour deposition (MOCVD) were used for measurements. Details of the sample preparation are given elsewhere28. SrRuO3, which was indexed by a pseudo-cubic notation like hklpc here, was grown by a RF-magnetron sputtering method reported in our previous paper29. For applying an electric field to this film, Pt top electrodes 100 μm in diameter were fabricated on the PZT films by electron-beam evaporation.Pb beam line, BL15XU, of SPring-8, Japan.To determine the structural characterisations of the PZT films before applying an electric field, high-resolution X-ray diffraction (HRXRD) and 2In-situ observation of the change in lattice parameters and domain structure with an applied electric field was carried out at the BL13XU at SPring-8 for Pt/PZT/SrRuO3 capacitors. To acquire the electric field-induced changes in diffraction data, an incident beam with photon energy of 12.4 keV was focused down to a few micrometres in width and height using a two-dimensional focusing refractive lens and irradiated on the Pt top electrode with the help of a fluorescent X-ray detector. We applied pulse voltages with a magnitude of 10 V and monitored the changes in 400 and 004 diffraction peaks. A positive 10-V square pulsed voltage 200 ns in width was applied to the capacitor to obtain sufficient poling treatment prior to measurement. The 200-ns square pulsed electric field was applied using a pulse generator  and FCE-HS3 . Time-resolved X-ray diffraction was measured using a high-speed avalanche photodiode detector (APD). The pulse height generated by a time-to-amplitude converter (ORTEC 567), for which X-ray pulse measured by the APD detector was used as a start signal and pulse signal synchronized to the electric pulse applied to the PZT films was used as a stop signal, is canalized by a multi-channel analyser. This technique cannot detect more than one photon for one period, so that properly attenuated X-rays were irradiated. No remarkable fatigue was confirmed during measurements (see Fig. 30.see Fig. . DetaileSupplementary Information


Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. Bordetella pertussis uses the BvgAS sensory transduction two-component system to regulate production of its virulence factors. BvgS serves as a model for a large family of multidomain bacterial sensor kinases. B. pertussis is virulent when BvgS functions as a kinase and avirulent when it switches to phosphatase activity in response to modulating signals. Understanding the molecular regulation of those proteins might lead to new antibacterial strategies. Here, we show that the linker regions between the perception and the enzymatic domains shift between distinct states of conformation in an alternating manner in response to signals and that their antagonistic changes control sensor kinase activity. These linker regions and mechanistic principles appear to be conserved among BvgS homologues, irrespective of the presence or absence of an intervening domain between the perception and the enzymatic domains. This work has thus uncovered general molecular mechanisms that regulate activity of sensor kinases in the BvgS family.The whooping cough agent  Bordetella pertussis is the agent of whooping cough, an acute respiratory disease. Colonization of the human respiratory tract requires the production of virulence factors whose expression is coordinately regulated by a sensory transduction two-component system (TCS) called BvgAS. TCSs are widespread in bacteria and involved in regulating various cellular processes, including metabolism, mobility, bacterial development, and virulence. Perception of a specific signal by the sensor kinase leads to autophosphorylation of the kinase domain and transfer of the phosphoryl group to the response regulator (–egulator –3.+) phase. Under standard culture conditions, i.e., at 37°C and without ligand, BvgS is in a kinase mode. In the laboratory, BvgS shifts to a phosphatase mode upon perception of modulators such as MgSO4 and nicotinate or derivatives or under conditions of nutrient restriction or low temperature. This sets the bacteria in the avirulent, Bvg− phase  domains for signal perception , 10. EacTwo linkers, called linker 1 and linker 2, connect the VFT domains to the PAS domain and the PAS domain to the DHp domain, respectively . Found i12–Both linker 1 and linker 2 can form two-helix coiled coils. In canonical coiled coils, the two parallel α helices coil around one another in a left-handed manner. They are characterized by heptads of amino acid residues that form two helical turns, denoted by “abcdefg” letters . At the Signal transduction in BvgS proceeds by changes of the conformation and the dynamics of the various segments of the protein. The kinase state is characterized by great dynamics of the membrane-distal VFT1 domains, linker 1 adopting a coiled-coil conformation, the PAS domains forming a dimer, and linker 2 being dynamic , 19. Thi20–BvgS serves as a prototype for a family of more than 11,000 VFT-containing sensor kinases, some of which are found in major bacterial pathogens , 10. A sΔR1, BvgSΔR2, BvgSΔR3, and BvgSΔR4, in which linker 1, the PAS domain, and linker 2 of BvgS were replaced with the X linkers of four PAS-less BvgS homologues whose lengths differed by multiples of 7 residues  activity levels with fhaB-lacZ, indicating that the recombinant strain is in an intermediate, Bvgi phase  mode under basal conditions.Here, we constructed a new chimera called BvgS BvgSΔR2 . We char fusions  positivenditions . At the gi phase  and C. Areporter . No chanal state . Thus, B4, another chemical modulator  by the use of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) markedly reduced the kinase activity  with Ala generated BvgSΔ65–5A, which is locked in the kinase mode but with a lower maximum activity than that of modulated BvgSΔ65  analyses were performed after the positively charged region. Residues 574 to 595 were individually replaced by Cys, and an oxidative treatment was performed to favor in vivo formation of intradimer S-S bonds  or in the phosphatase mode (positions 593 and 594). We checked the production and stability of the latter two in B. pertussis membrane extracts to dismiss the possibility that the proteins were proteolyzed in vivo. Both were properly produced  or after the addition of 5 mM chloronicotinate (CN5) (+). The monomeric and dimeric forms of BvgSΔ65t are denoted “M” and “D,” respectively. The “a” to “g” positions of the two-helix coiled-coil heptads are indicated below each substitution. The proportions of dimers, determined as indicated at the top left, are given at the top of each lane. Download FIG S1, TIF file, 0.8 MB.Cys-scanning analyses of linker X of BvgSCopyright © 2018 Lesne et al.2018Lesne et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02052-17.2FIG S2 B. pertussis were performed. The ΔbvgA strain is shown as a control to show the abundance of BvgS in an avirulent strain. The loading control was an unidentified B. pertussis protein fortuitously recognized by the antibodies (indicated with an asterisk). Note that in several instances spontaneous formation of an S-S bond between monomers resulted in the presence of dimers (denoted “D”). (A) Cys variants of BvgSΔ65. (B) Size variants of BvgSΔ65. (C) Size variants of BvgSΔ70A and BvgSΔ70B. (D) Variant of BvgSΔ72 LLAAA harboring substitutions that destabilize interhelix interactions. Download FIG S2, TIF file, 0.8 MB.Detection of the inactive BvgS variants. Immunoblot analyses of BvgS variants in membrane extracts of Copyright © 2018 Lesne et al.2018Lesne et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Δ65 as found in chimeras BvgSΔR1 to BvgSΔR4, we deleted one residue immediately after the TM segment, yielding BvgSΔ64. We similarly constructed size variants BvgSΔ62, BvgSΔ63, and BvgSΔ66 and tested the effects of those modifications on activity  are indicated with dashed lines, blue for favorable ionic interactions and red for unfavorable interactions. Potential intrahelix ionic interactions between i and i+3 or i and i+4 residues adopting “b,” “c,” or “f” coiled-coil positions are indicated on one monomer, using black dotted lines. Download FIG S3, TIF file, 1.5 MB.Intra- and interhelical ionic interactions for BvgSCopyright © 2018 Lesne et al.2018Lesne et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Δ65 and screened for functional sequences. In addition, we modified their length to obtain the same heptad periodicities as in BvgSΔ65 and BvgSΔ64  simulations for each. The simulations were analyzed to determine the features of the linkers that are functional or partially functional in the context of our BvgS platform, i.e., that cause shifts of activity upon modulation. For the BvgSΔ65, BvgSΔ65 Se, and BvgSΔ70C linker sequences, the levels of helical stability notably differed between the two sets of simulations  are indicated with dashed lines, blue for favorable ionic interactions and red for unfavorable interactions. In BvgSΔ72C Inter, three putative favorable interhelix interactions were replaced by unfavorable interactions to weaken coiled-coil formation along the K register. Potential intrahelical ionic interactions between i and i+3 or i and i+4 residues adopting “b,” “c,” or “f” coiled-coil positions are indicated on one monomer in each case, using black dotted lines. Additional intrahelix interactions in BvgSΔ72C Intra compared to BvgSΔ72C LLAAA are indicated with green dotted lines. The substitutions introduced in BvgSΔ72C Intra were aimed at adding putative intrahelix interactions to stabilize the α helices and thus to facilitate the interconversion. Download FIG S4, TIF file, 2.3 MB.Intra- and interhelical ionic interactions for BvgSCopyright © 2018 Lesne et al.2018Lesne et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 575E, A579R, K582E, I589E, and I593R were combined to strengthen the α helices by increasing the number of potential intrahelix ionic interactions  in the initial set of sequences were very rare. Linker X of BvgSSequence alignments showed a high level of conservation in the two heptads that precede the DHp domain . The LLA571 in BvgSΔ65 and R570 in wt BvgS) shown to be critical for regulation was very often found at the “d” position in the second heptad of the X linkers composed of at least 4 heptads. Overall, conserved features of linker X in the family support our model that its composition generates the coiled-coil antagonism that regulates the output activity of the system.In the first heptads of the X linkers, a high density of charged residues was generally conserved, as were a few residues at both interfacial and noninterfacial positions of register K. In particular, a positively charged residue  or the Arg570-to-Ala variant in linker 1 of BvgS . In the first moiety of linker X, a moderately stable coiled coil is predicted along register K, with a suboptimal interfacial motif that is compensated for by favorable inter- and intrahelical ionic bonds. Removing interhelical ionic interactions along register K in that region favors the phosphatase mode. Conversely, enhancing coiled-coil formation along register K by strengthening the interface favors the kinase mode, as shown by the kinase-locked phenotypes of the LysWe propose that the antagonism between the two coils in the PAS-less BvgS homologues forms the basis for regulation: coiled-coil formation along register K prevents coiled-coil formation along register P and vice versa, and signal perception triggers interconversion. The dynamics of the last portion of linker X is conducive to kinase activity, which has been proposed to require dynamic asymmetry of the DHp helices in model sensor kinases, while the phosphatase state corresponds to a more rigid state , 22. By Δ65 chimera displays inverted regulation; i.e., modulators cause a strong increase of kinase activity. In wt BvgS, modulator binding to the periplasmic domain causes a small piston motion that pulls one residue of linker 1 into the membrane, thus disrupting the first coiled coil  locked phenotypes might be due to the accommodation of the two coiled-coil registers by loose α helices  liquid medium at 37°C under conditions of agitation. To construct BvgS chimeras devoid of the PAS domain, synthetic gene portions  coding for the junction between the TM segment and the DHp domain of BvgS homologues were introduced by BglII-XbaI cassette exchange in pUC19mpla , and the first part of the BvgS kinase. Cassette exchanges were performed in pPORVPH , encompassing the VFT, TM, linker X, and HK domains as previously described  were used to determine the effect of Cys substitution on BvgS activity.The disulfide (S-S)-mediated cysteine cross-linking-mediated (Cys-scanning) analyses were performed in escribed . In this2, respectively. The systems were solvated with simple point charge (SPC) water  water  in a rhoemployed , using iemployed . van deremployed . Equatioemployed . The proemployed . Data exPF00497  and PF00512 (His kinase A domain) domains were parsed, considering only hits with an E value of <0.001 for both domains. Using a customized Python script, we selected the sequences containing two VFT domains, which resulted in selection of 8,218 TCS sequences. They were divided in two groups, those without PAS domain  and those with a single PAS domain , using modified versions of the script mentioned above. TM domain predictions were done for all those sequences, and a few sequences were discarded because of the absence of a clearly predicted TM segment.hmmsearch output files consisting of all predicted proteins identified from the NCBI nonredundant database (release of September 2017) containing both Sequences devoid of a PAS domain were then reduced to a fragment starting from the end of the predicted TM segment and extending to the end of the HisKA motif, yielding 740 unique sequences of linker X due to high redundancy of this part of the sequences. Sequences containing a single PAS domain were similarly reduced to the end of the HisKA motif, yielding 767 unique sequences, after discarding atypical sequences in which linker 1 was longer than 34 residues. From this set, the linker 1 sequences were extracted for the alignment from the end of the predicted TM segment to the last residue before the PNP motif that initiates the PAS domain. Similarly, the linker 2 sequences included the residues found after the DIT (Asp-Ile-Thr) motif that terminates the PAS domain and before the beginning of the DHp domain.http://weizhongli-lab.org/cdhit_suite), with an 80% cutoff for the linker X sequences and a 50% cutoff for the linker 1 sequences. The 30 most highly populated clusters of linker X sequences and the 15 most highly populated clusters of linker 1 sequences were further analyzed. Sequences were divided into heptads along register P based on a prior coiled-coil prediction using PCOILS (https://toolkit.tuebingen.mpg.de). Heptad-based manual alignment of different clusters was carried out using Jalview (The two sets of sequences were clustered using CD-HIT ( Jalview , generatfhaB-lacZ fusion but no activity with the ptx-lacZ fusion, were described as having low kinase activity, while the chimeras yielding no reporter activity with either fusion were described as being in the phosphatase state.β-Galactosidase assays were performed as described previously  with thrB. pertussis, the bacteria were lysed, and the membrane proteins were harvested by ultracentrifugation before immunodetection of BvgS was performed as previously described (For the detection of inactive BvgS variants in escribed . For theescribed .


Since Ni-based single-crystal superalloys are anisotropic materials, their behavior in different crystal orientations is of great interest. In this study, the yielding behavior in both tension and compression for  Ni-based single-crystal superalloys are used as blade material in gas turbines and aero engines thanks to their excellent resistance to mechanical and chemical degradation at elevated temperatures ,2. By uset al. [It is well recognized that Ni-based single-crystal superalloys exhibit a number of surprising mechanical properties, including an increase in yield strength with temperature, strong orientation dependence of the yield stress and a tension/compression asymmetry ,4,5,6. Tet al. , the croet al. [Sometimes also a serrated yielding behavior is in progress during plastic deformation. Many metallic materials show a serrated flow, also known as the Portevin-Le Chatelier (PLC) effect. The reason for this behavior is the interaction between diffusing solute atoms and moving dislocations, more commonly known as dynamic strain aging (DSA). It is well known that the DSA phenomena may lead to a serrated yielding effect and in the literature one can find that DSA occur in superalloys at temperatures from 260 to 800 °C . Researcr et al.  reportedStudies concerning the behavior of −5 s−1. The samples were carefully aligned to prevent buckling. The tests were interrupted when approximately 1% plastic strain was achieved, and the specimens were then unloaded, cooled rapidly to room temperature (RT) and removed from the test machine. After the tests, all specimens were investigated by stereomicroscopy before they were cut parallel to the loading direction for further investigation by scanning electron microscopy (SEM) in a Hitachi SU70 SEM. The SEM samples were prepared by grinding and mechanical polishing, but no samples were etched. Acceleration voltages from 10 to 20 kV were used during the SEM investigation. Most images were taken using backscattered electrons and channelling contrast. Electron backscatter diffraction (EBSD) analysis was performed using an Oxford Instrument Nordlys detector.In this study the Ni-based single-crystal superalloy MD2 with chemical composition Ni, 5.1 Co, 6.0 Ta, 8.0 Cr, 8.1 W, 5.0 Al, 1.3 Ti, 2.1 Mo, 0.1 Hf and 0.1 Si was investigated. The material was solution heat treated at 1275 °C for 8 h followed by a two-stage aging process with 3 h at 1100 °C and 24 h at 850 °C. Test specimens were machined from cast bars and the deviation from the ideal orientation was less than 2° for the The results from tensile and compressive tests at 500 °C for the three crystallographic directions i.e., no significant asymmetry was found for these directions. In addition to the tension/compression asymmetry, the As expected, the The results can be compared to another study  where thAt both temperatures, the greatest tension/compression asymmetry in yield strength was found for the Specimen investigation by stereomicroscopy showed clear crystallographic deformation bands stretching over the Further investigation by SEM, using backscattered electrons, also showed clear deformation bands in the microstructure for the i.e., dislocations that propagate through the crystal. Hence, the dislocation density within the deformation bands is higher compared to the less deformed microstructure. Deformation bands in EBSD was performed to further examine the orientation variations between the deformation bands and the less deformed γ/γ'-microstructure. The mapping showed very small orientation variations, up to 1°, between the deformation bands and the less deformed microstructure. This means that twinning of the microstructure is not present within the deformation bands. In terature , and in i.e., the deformation bands. This can explain the serrated yielding behavior showed by the Thus, the us study , no serret al. [Hence, DSA in combination with highly localized deformation bands is one reason for the serrated yielding behavior of the r et al. , who conTo conclude this study, it is very clear that the