Recent metagenomic studies in insects identified many sequences unexpectedly closely related to plant virus genes. Here we describe a new example of this kind, insect R1 LINEs with an additional C-terminal domain in their open reading frame 2. This domain is similar to NTPase/helicase (SF1H) domains, which are found in replicative proteins encoded by plant viruses of the genus Tobamovirus. We hypothesize that the SF1H domain could be acquired by LINEs, directly or indirectly, upon insect feeding on virus-infected plants. Possible functions of this domain in LINE transposition and involvement in LINEs counteraction the silencing-based cell defense against retrotransposons are discussed. RNA helicases are RNA-binding proteins that utilize the energy derived from the hydrolysis of NTPs to unwind double-stranded (ds) nucleic acids. They are classified into six superfamilies designated SF1H to SF6H on the basis of conserved helicase motifs  and movement protein  and Turnip vein-clearing virus (Z29370) as queries for TBLASTN search at NCBI, we found two significant matches to the encoded amino acid sequences in the transcriptome shotgun assembly databases of Heliconius melpomene and Ostrinia nubilalis . Amazingly, our further analysis revealed more than 40 significant matches to the encoded amino acid sequences in the whole-genome shotgun assemblies of Plutella xylostella chromosomes (data not shown). The genome-integrated SF1H sequences share a similar level of amino acid sequence identity with different tobamoviruses. In particular, ToMV SF1H had 36% identity  with P. xylostella, 30% identity with H. melpomene , and 32% identity  with O. nubilalis sequences.Using replicative SF1H sequences of tobamoviruses H. melpomene, O. nubilalis, and P. xylostella was used as a query for TBLASTN search of virus sequence database, the most significant similarity of the consensus sequence was found to tobamoviral and related NTPase/helicase replicative domains of other plant viruses of the family Virgaviridae.When a consensus sequence based on an alignment of three tobamoviral-like SF1H domains encoded by H. melpomene, O. nubilalis, and P. xylostella sequence regions encoding the tobamovirus-like SF1H domains were found within long interspersed elements (LINEs). LINEs represent a class of non-LTR  retrotransposons widely spread in eukaryotic genomes. The majority of functional LINEs code for ORF1 and ORF2 proteins, the latter comprises several functional domains, which specify retropositional activities such as reverse transcriptase, endonuclease and, in some cases, RNase H . The most C-terminal region in the LINE ORF2 is known to tolerate insertions of foreign protein domains. For example, a family of plant LINEs was found to code for the ORF2 protein with an additional C-terminal RNase H domain of archaeal origin; moreover, this domain appeared autonomously active . We further analyzed whether such LINEs could be found in other Lepidoptera families and other insect orders. Nucleotide and amino acid consensus sequences of SF1H based on the alignments of H. melpomene, O. nubilalis, and P. xylostella were used as queries for searches of nucleotide and translated nucleotide databases, respectively.The three insect species for which SF1H-like domain-encoding LINEs were found, Operophtera brumata (the family Geometridae), Polyommatus icarus (Lycaenidae), Samia ricini (Saturniidae), Antheraea pernyi (Saturniidae), Manduca sexta (Sphingidae), Spodoptera litura (Noctuidae), Melitaea cinxia , Plodia interpunctella , and Yponomeuta evonymellus (Yponomeutidae)  and Danaus plexippus , no SF1H-like domain-encoding LINEs were found. This finding indicated that not all lepidopterans contain LINEs of this type. On the other hand, the presence of these LINEs in different branches of the Lepidoptera phylogenetic tree including early diverged families Plutellidae and Yponomeutidae  databases. In addition to the three initially identified species, the SF1H-like domain-encoding LINEs were found in nine other species, namely, ) Figure . Importae Figure  demonstrFurther, we analyzed species of three orders most closely related to Lepidoptera. Overall, for the order Hymenoptera  genomic sequences were analyzed in 57 species, for 27 of which the complete or very extensive genome information was available; for the order Diptera (flies), for 32 species (13 with complete or well-represented genomes); and for the order Trichoptera (caddisflies), for four species  but identified in representatives of the families Saturniidae and Sphingidae, which, together with Bombycidae, belong to the superfamily Bombycoidea  diverged early in the Lepidoptera evolution, indicating that the SF1H domain could be acquired by LINEs in an ancestor that existed before the divergence of Lepidoptera lineages Figure . On the a Figure . Even moDrosophila are known to be subject of frequent transposition and elimination by selection  and several species of more recently diverged families were found to be in the range expected for vertically inherited retrotransposons diverged from a last common ancestor at the estimated time of Plutellidae divergence from other analyzed families , the mRNA coding for the SF1H domain of SF1H-containing LINE was found among expressed genes at least in the egg, late larval, and pupal stages, future experiments on heterologous expression of native and mutant SF1H LINEs can shed light on possible influence of the SF1H domain on LINE mRNA synthesis and stability. Another experimental line can include investigation of the helicase activity of LINE-encoded SF1H domain on different substrates, primarily LINE replication intermediates.This hypothesis finds some support in our experiments, in which the s Figure . Such cos Figure , 2014; hs Figure . NeverthConsidering the future directions of the experimental elucidation of the significance of viral helicase domains acquired by retrotransposons, we envisage studies of functional properties and expression of retrotransposon-encoded SF1H domains in insect organisms and cultured insect cells. However, it should be stressed that the data presented in this paper can represent only the tip of the iceberg, since preliminary database searches reveal multiple occurrences of so-far uncharacterized SF1H-like sequences in the genomes of non-lepidoteran insects. Additionally, cucumoviral and pomoviral helicase sequences were revealed in close proximity to plant LTR-type retrotransposons. Therefore, further extensive bioinformatics analysis can shed light on the whole picture of viral SF1H invasion of eukaryotic genomes, while functional studies of LINE-encoded SF1H domains will clarify their functional significance in the context of insect retrotransposons.AS and SM designed and planned the research. EL and AL performed the experiments. AS and SM performed database searches. NV performed phylogenetic analysis. AS, NV, and SM wrote the manuscript. All authors reviewed the manuscript.Sequence bank mining and phylogenetic analysis was performed by NV at the Engelhardt Institute of Molecular Biology with support of the Russian Foundation for Basic Research (grant 13-04-01678). All other work was performed by EL, AL, AS, and SM at Moscow State University with financial support of the Russian Science Foundation (grant 14-14-00053).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition.The X-ray structure of protease-cleaved  Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved  ECAM was initially overexpressed in E. coli BL21(DE3) cells and subsequently in T7 Express Crystal Competent E. coli cells  using an inducible T7 promoter with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) as the inducer. Bacteria expressing ECAM were grown in lysogeny broth; selenomethionine-labelled ECAM was obtained using M9 minimal medium supplemented with 50 mg l−1 selenomethionine and 20 mg l−1 of each of nine essential amino acids (excluding methionine). Cells were grown at 37°C to an OD600 of 0.6, protein production was induced by the addition of 1 mM IPTG and the cells were grown for a further 6 h. The cell pellet was collected by centrifugation at 4400g for 15 min and the cells were resuspended in binding buffer  and lysed by sonication with 1 mg ml−1 lysozyme in the presence of protease inhibitors . Cell debris was removed by centrifugation at 46 000g for 30 min at 4°C. The cell supernatant was then loaded onto a HisTrap HP column  and the bound protein was eluted with elution buffer  using a linear gradient increasing from 10 to 500 mM. Fractions containing ECAM were pooled and dialysed overnight at 4°C into 50 mM Tris, 200 mM sodium chloride pH 7.5 and run on a Superdex S200 gel-filtration column . Central fractions from the peak were combined and concentrated using a 100 kDa molecular-weight cutoff centrifugal concentrator.The gene for ECAM, 2.2.M Tris, 200 mM NaCl pH 7.5 on ice for 5 min before being loaded onto a Superdex S200 gel-filtration column . The two major peaks from gel filtration were concentrated to 16 mg ml−1 separately using 100 kDa molecular-weight cutoff centrifugal concentrators and used in crystallization trials. Several hundred crystallization conditions were tested, including the JCSG-plus, MIDAS and Morpheus screens (Molecular Dimensions), for both concentrated peaks. A Cartesian Honeybee 8+1 (Harvard Bioscience) robot was used with 96-well plates, dispensing 0.5 µl reservoir solution and 0.5 µl protein sample. Subsequent scaled-up crystal growth was performed using 2.5 µl reservoir solution and 2.5 µl protein sample. The initial crystal was grown in conditions consisting of 0.1 M potassium chloride, 0.1 M HEPES, 25% Sokalan CP 7 pH 7.0, and upon optimization the pH was adjusted to 7.5 for larger crystal growth. Crystals were grown using equal volumes of protease-cleaved ECAM and reservoir solution using sitting-drop vapour diffusion, with crystals appearing after 2 d at 16°C for the second fraction and after two weeks for the first fraction. Cryoprotection was optimized with a 3:2 ratio of xylitol-saturated reservoir solution to reservoir solution. Crystals were briefly soaked and flash-cooled in liquid nitrogen for data collection. The best diffraction resolution obtained was 3.8 Å, and molecular replacement with methylamine-activated α2M (PDB entry 4acq) was unsuccessful, most likely as the sequence identity with the human homologue was low (12%) and owing to the difference in domain orientation between the structural models  and the purification and crystallization screens were repeated using selenomethionine-labelled protein. As repeating the previous protocol with selenomethionine-labelled protein was unsuccessful, in situ proteolytic cleavage screening was performed using porcine elastase. Successful crystallization was achieved using a 1:100 ratio of porcine elastase to selenomethionine-labelled ECAM. Crystallization was successful in the same condition as used previously, with the crystal having a similar appearance and the same space group as previous unlabelled crystals. These crystals diffracted to 3.65 Å resolution and phases were obtained using single-wavelength anomalous diffraction (SAD).Purified ECAM was reacted in a 1:1 molar ratio with porcine elastase  in 50 mK edge (λ = 0.97939 Å) using a PILATUS 6M detector. A high-redundancy SAD data set was processed and scaled using XDS and AIMLESS from the CCP4 suite of programs . This yielded interpretable, low-resolution maps in which density corresponding to secondary-structure elements and larger amino-acid side chains was visible. Initially, the α-helical TED domain was built in Coot using idealized α-helical sections . Statistics for data collection, experimental phasing and refinement are presented in Table 1Data were collected for ECAM crystals on the I02, I03 and I24 beamlines at Diamond Light Source, Didcot, England at 100 K at the Se 3.3.1.M potassium chloride, 0.1 M HEPES, 25% Sokalan CP 7 pH 7.0 with cleaved ECAM. In order to obtain phase information, we attempted to repeat this process with selenomethionine-labelled ECAM, but this failed to yield crystals. However, an alternative strategy of in situ proteolysis and crystallization with selenomethionine-labelled ECAM was successful. This method yielded crystals that diffracted to 3.65 Å resolution. Upon completion and validation of the model of protease-activated ECAM, the number of Ramachandran outliers remaining was appropriate for a crystal structure with a resolution of 3.65 Å. Although the Rmerge and Rp.i.m. values were high, this can be explained by the highly redundant data set used; with a CC1/2 of 0.675 in the highest shell, these data used were judged to be acceptable . Elastase-cleaved ECAM  adopts a conformation similar to that of methylamine-activated human α2M  but distinct from the unactivated form of SaA2M . Despite the low sequence identity between human α2M and ECAM (12%), the r.m.s.d. between Cα atoms for these proteins is 14.1 Å, while that for SaA2M and ECAM, which share 82% sequence identity, is 22.1 Å. Most notably, in the structure of the protease-cleaved ECAM, interactions between the TED and the CTMG domain, which protects the thioester bond in unactivated SaA2M, are not present. Instead, the thioester region of TED is solvent-exposed and faces the expected location of the attacking protease, as it would be positioned when cleaving the bait region of ECAM . Electron density for elastase, in addition to that for MG domains 1, 2, 3 and 7, was absent in 2Fo − Fc maps of cleaved ECAM, as was electron density for 20 amino-acid residues (Arg923–Leu942) within the bait region. Analysis of elastase-cleaved ECAM by SDS–PAGE and mass spectrometry confirms that MG domains 1, 2, 3 and 7 are absent from the crystallized protein . Two bands at 90 and 75 kDa on SDS–PAGE have similar peptide coverage, encompassing the same domains . However, the reason for the difference in apparent molecular weights between these two species is not known.Similar to the domain architecture of native SaA2M, uncleaved ECAM consists of ten MG domains, with a largely disordered bait-region domain found within MG8, a TED that houses the reactive thioester bond and a CUB domain Fig. 1a. ElastAM Fig. 1b adopts2M Fig. 1c but di2M Fig. 1d. DespiAM Fig. 1b. Elect3.2.et al., 1979et al., 2012a). Critically, the region of the CTMG domain that is involved in BRD binding substantially overlaps with the region of the CTMG domain that forms the binding interface with the TED in the uncleaved form of the protein . The buried surface area between the CTMG domain and TED in SaA2M is 1972 Å2, with three hydrogen bonds between these domains, whereas the buried surface area between the elastase-cleaved BRD and the CTMG domain in ECAM is 1438 Å2, with five hydrogen bonds between the domains. In the protease-cleaved ECAM, hydrogen-bond formation between residues of the CTMG domain and the cleaved bait region involves the highly conserved RDDR and EXMY motifs . The movement of the TED domain shows an overall shift of 36 Å, with the MG6 domain moving by 50 Å and with both the MG6 and TED arms hugging the position at which the attacking protease would be located to cleave the BRD . In addition to these global conformational changes, protease cleavage leads to localized changes in the environment of the thioester bond that are likely to lead to its activation. In the unactivated SaA2M structure the conserved methionine and tyrosine side chains (Met1625 and Tyr1626 in SaA2M) from the CTMG domain form part of the hydrophobic pocket at the interface with the TED that has been shown to be important in maintenance of the thioester bond . As there are no contacts present from the CTMG domain providing a hydrophobic pocket to protect the thioester from hydrolysis by water, the thioester bond could be cleaved and the deaminated glutamine (Gln1190) would be converted to a glutamic acid. Within our protease-activated ECAM structure the distance between the S atom of Cys1187 and the Cγ atom of Gln1190 is 4.6 Å, indicating that the thioester bond may not be intact . However, we cannot rule out the possibility that there is a mixed population of molecules, some of which possess an intact thioester bond. Owing to this ambiguity, we have represented the thioester without a covalent bond between Cys1187 and the Cγ atom of Gln1190 and have also omitted the O atom from the deaminated glutamine that would be formed on hydrolysis of the thioester bond.We propose that the loss of the interaction of the TED with the CTMG domain is sufficient to enable both the large TED movement observed in the cleaved ECAM structure relative to the unactivated SaA2M structure and the exposure of the thioester bond .. DOI:


Charged ferroelectric walls, which are energetically unfavourable in general, were found to be mysteriously abundant in hybrid improper ferroelectric 3Ti2O7 crystals. From the exploration of antiphase boundaries in bilayer-perovskites, here we discover that each of four polarization-direction states is degenerate with two antiphase domains, and these eight structural variants form a Z4 × Z2 domain structure with Z3 vortices and five distinct types of domain walls, whose topology is directly relevant to the presence of abundant charged walls. We also discover a zipper-like nature of antiphase boundaries, which are the reversible creation/annihilation centres of pairs of two types of ferroelectric walls  in 90° and 180° polarization switching. Our results demonstrate the unexpectedly rich nature of hybrid improper ferroelectricity.Charged polar interfaces such as charged ferroelectric walls or heterostructured interfaces of ZnO/O and LaAlO Charged ferroelectric domain walls show promise for two-dimensional conduction, but their abundance within 3Ti2O7 crystals is poorly understood. Here, Huang et al. discover topology related domain structures in such materials, which reveal the rich nature of hybrid improper ferroelectricity. Directional-variants result in domains with different directional order parameters. The role of APBs16181920213B2O7 3273Ti2O7 crystalsHybrid improper ferroelectricity (HIF), a phenomenon involving polarization induced by a hybridization of two non-polar lattice instabilities, offers great promise towards the realization of room-temperature multiferroism27282930272829312.55Sr0.45Ti2O7  and Ca3Mn1.9Ti0.1O7  single crystals with in-plane polarization along the pseudo-tetragonal [110] directions3304 × Z2 domain topology with Z3 vortices is responsible for the presence of abundant charged FE DWs in CSTO. In addition, we have also investigated the kinetics associated with polarization switching in CSTO, understanding of which is crucial for developing precise control of conducting FE walls.To unveil the origin of these abundant charged FE DWs, we have explored the complete connectivity of DWs in Ca3B2O7: First, the BO6 octahedral in-phase rotations are either clockwise (the sign of the rotation is +) or anticlockwise (−) about a [001]T direction (denoted as a0a0c+ in the Glazer notation2+ mode), and second, the BO6 octahedral tilting occurs about two <110>T axes, that is, apical oxygen-motions displace towards the 1st to 4th quadrants (denoted as a−a−c0 or the X3− mode) with respect to the high-symmetry tetragonal I4/mmm  structure. Below the phase transition, the X3− mode adopts one of the four tilts (1–4) accompanying the X2+ mode with + or − rotations into a combined distortion pattern of a−a−c+ having eight degenerate states, which we label as 1±, 2±, 3± and 4±  with a clockwise rotation (black curved arrow) to form a 1+ state. A trilinear coupling among the X2+ mode, X3− mode and the polar Γ5− mode (A-site displacement) yields four FE polarizations parallel or antiparallel to the two <110>T tilting axes33− and X2+) changing signs. These four polarization directional variants with twofold degeneracy form the basis of the Z4 × Z2 domain structures in the HIF A3B2O7. 3B2O7, but behave hidden in the PFM images as the two domains give the same piezo-responseT direction using superlattice peaks g1±=±3/2T parallel to the polar axis within a single FA domain. Three domains (i–iii) in 3 vortex. Note that the relative polar ±aorth directions can be identified from the related electron diffraction patterns but the absolute polarization direction cannot be. Thus, once the polarization direction is chosen for one domain, then the polarization directions in other domains can be fully assigned without ambiguity. Evidently, the existence of APBs remains unchanged even if the assignment of the polarization direction is reversed. 3 vortex also suggests the existence of pure rotation (a0a0c+)-driven DWs and pure tilting (a−a−c0)-driven DWs. We define a tilting-type FEt DW (rotation-type FEr DW) as the wall between adjacent FE domains having opposite a−a−c0 tilting (a0a0c+ rotation) but identical a0a0c+ rotation (a−a−c0 tilting). The structural details of FEt and FEr DWs are shown in 3 vortex in r (red-dotted), FEt DWs (red-solid) and APB (green line).Polarized optical microscope images on CSTO and CMTO crystals both clearly exhibit orthorhombic twins, that is, orthorhombically distorted ferroelastic (FA) domains. In addition, in-plane PFM studies show the intriguing FE domains comprising abundant meandering head-to-head and tail-to-tail charged DWsg1± contributed from the FA(i) domain marked by red and blue circles in 3-vortex network is clearly visible within the FA(i) region. g1− spot (blue-circled) and the schematic (3 vortices is linked by an APB (green line). Boundaries between the 3− (pink) and 3+ (blue) domains form broad contrast walls, identified as FEr DWs (red-dotted lines of chematic  shows a 1amA2) as Ca3Ti2O7 at 300 K. T surface, distinct from the prototypical straight FA DWs  in CSTO. Our DF-TEM images  between states in the same rotation, for example, the 1+ and 2+ or 3− and 2− (blue-dotted lines in tr) DWs between, for example, the 2+ and the 3− states , whereas CMTO shows a 64% population. The low population in CMTO is likely related with the existence of irregular FA domains in CMTO. Note that although an energy hierarchy may exist, we do observe experimentally and theoretically all five kinds of DWs.We also employed the phase-field method 333Mn2O7, nucleated from the Acaa (space group #68) phase1amA2 domains from the Acaa matrix is shown in The energy diagram for the HIF A diagram  are showIn-situ poling results on CSTO using a DF-TEM technique unveil intriguing domain switching kinetics, which can be understood in terms of the creation and annihilation of Z3 vortex-antivortex (V-VA) pairs. In-situ poling is achieved utilizing fast positive charging40in-situ poling process, DF-TEM images are obtained immediately after defocusing the electron beam, before the charges are completely dissipated (on the order of 30 min). 3 V-VA pair (cyan and blue circles) within one FA domain behaving coherently with in-situ poling. In-situ poling at a 5 o'clock position near the crystal edge induces a direct 180° polarization reversal of a 4− (light green) domain to a 4+ (yellow) domain ; FEt DWs with a larger energy than that of FEr tend to be pinned at the original APB location, whereas FEr DWs tend to be mobile. This observation is in accordance with the energy hierarchy of FEr≤FEt≤APB discussed earlier.) domain , which i) domain . Eventuatr DW and the other FAt DW 3Ti2O7. These unexpected discoveries of the role of APBs and the domain topology relevant to the presentence of abundant conducting DWs should be further investigated for deeper understanding and nano-engineering of the domains and DWs in hybrid improper FEs.We observe a direct 180° (90°) polarization switching, instead of going through an intermediate 90° (180°) polarization state in the ed walls  leads to3-xSrxTi2O7 (Ca3Mn2-xTixO7) feed rods, stoichiometric CaCO3, SrCO3 and TiO2 (MnO2) were mixed, ground, pelletized and sintered at 1,350–1,550 °C for 30 h. Substituting Sr into the Ca site in Ca3Ti2O7 induces the reduced size of FA domains suitable for TEM studies. It was very difficult to grow high-quality single crystals of pure Ca3Mn2O7, but the slight substitution of Ti into the Mn site of Ca3Mn2O7 stabilizes crystal growth without changing the relevant physics of Ca3Mn2O7. Crystals are highly cleavable, and were cleaved in air for optical microscopy observation. Transparent amber coloured CSTO single crystals and non-transparent dark blue coloured CMTO present a similar polar orthorhombic symmetry 330a−b)/(a+b)*100%, ∼0.08 %) than Ca3Ti2O7 (∼0.05 %). Cycling the CMTO sample temperature through Tc leads to a completely different irregular FA pattern, indicating that the domain formation is not simply due to pinning by disorder such as chemical defects or dislocations.Single-crystalline CSTO and CMTO were grown by using optical floating zone methods. For polycrystalline Ca3Ti2O7 (CTO), Ca2.55Sr0.45Ti2O7 (CSTO) and Ca3Mn1.9Ti0.1O7 (CMTO) single crystals (∼1 × 2 × 0.1 mm3 in size) by mechanical polishing, followed by Ar-ion milling and studied using a JEOL-2010F TEM. Note that one TEM specimen can include up-to-a-hundred FA domains for observations, and we have examined two CSTO, one CTO and two CMTO TEM specimens. Although the width of FA domains varies, our conclusion on the Z4 × Z2 domain structure with Z3 vortex patterns and five types of DWs is universal in all specimens that we have observed. We also found that the FA domain size depends little on various heat treatment conditions in both CSTO and CMTO. We observed vortex domains by DF-TEM imaging taking two diffraction vectors: (i) superlattice g1±=±3/2T=±orth spots, parallel to the polar axis in [001]T zone and (ii) superlattice g2±=±3/2T=±orth spots, perpendicular to the polar axis in T zone, 15° tilting from c-axis. 2+ spot. Non-FE structural boundaries can be visualized under this condition as the g2+ vector is perpendicular to the polar aorth-axis and the FE contribution is minimized. To avoid confusion with APBs, those boundaries observed in this condition without considering the polarization effect are named as ‘B-boundaries'. The appearance of ‘B-boundaries' is a result of symmetry breaking through the tetragonal-to-orthorhombic phase transition. They tend to show step-like features along the <100>T direction. Based on the domain switching kinetics shown in t DWs, so APBs and FEt DWs tend to be <100>T-oriented. Contrarily, FEr DWs show wavy features with no preferred orientation, because of the so-called ‘rotational compatibility conditions'g1+ spot, showing the domain contrast of neighbouring FA(ii) region. A full assignment of domain states and DW types in a CSTO crystal is shown in Specimens for DF-TEM studies were fabricated on CaT-oriented FEt DW between two neighbouring 1+ and 3+ domains, where the octahedral tilting (a−a−c0) may be fully suppressed if octahedral tilting changes across the DW by passing through the tetragonal central position  for the oxygen octahedral rotation around the x3 axis, and θi and Pi for the octahedral tilt and polarization component along the xi pseudocubic axis, respectively23To describe the distortion relative to the high symmetry phase with space group αij, αijkl, βij, βijkl, tijkl, d and γij are the coefficients of Landau polynomial, κijkl, δijkl and gijkl are gradient energy coefficients, cijkl is the elastic stiffness tensor, ɛij and Ei is the electric field given by Ei=−ϕi, with ϕ the electrostatic potential. Note that γij>0, and the term dϕ3(θ1P1+θ2P2) determines that  and  are parallel or antiparallel, dependent on the sign of ϕ3. The eigen strain is related to the order parameters through λijkl and hijkl are coupling coefficients. Here the coupling between polarization and strain is ignored, as the secondary order parameter polarization is always parallel or antiparallel to the octahedral tilt order parameter. The Landau polynomial is expanded based on group theory analysis384344454647κijkl and δijkl, that is, κiiii≪κijij, δiiii≪δijij  is used for the Brillouin-zone integrations. The calcium 3s, 3p and 4s electrons, Ti 3p, 3d and 4s electrons, and O 2s and 2p electrons are treated as valence states.Total energy calculations based on density functional theory within the generalized-gradient approximation given by the revised Perdew–Becke–Erzenhof parameterization for solids44I4/mmm structure (parent clamping approximation). The total elastic stiffness tensor, including the contributions for distortions with rigid ions and the contributions from relaxed ions, is also obtained by calculating the strain–stress relationsI4/mmm structure .Supplementary Figures 1-5The nucleation and growth of the ferroelectric A21am phases from the centrosymmetric Acaa matrix in Ca3Mn2O7 are demonstrated by the phase-field simulations. The initial domain structures consist of two variants of the Acaa phase, which are denoted by deep blue and yellow-green colors. The final domains are the eight variants of the A21am phase with the same color assignment as in the main text.A movie of the FEr domain wall motion in a Ca2.55Sr0.45Ti2O7 crystal after defocusing the electron beam of a transmission electron microscope, which corresponds to Fig. 5b to c (the size of the field of view is ∼ 2 μm x 2 μm).


The objectives of this study were to report performance and management data taken from a sample of UK dairy farms that have participated in the Agriculture and Horticulture Development Board Dairy Mastitis Control Plan (DMCP) and to identify important mastitis prevention practices that are not currently widely implemented. A total of 234 UK dairy herds were included in the study from which farm management and udder health data were collected. Herds were grouped according to their mastitis epidemiology and could be classed as (i) environmental dry period (EDP) , (ii) environmental lactation (EL), (iii) contagious dry period (CDP) or (iv) contagious lactation (CL). The results of this study showed that many mastitis-related management practices that are generally considered to be important were not widely performed. A better understanding of those practices not widely adopted by UK dairy farmers at present may aid practitioners in identifying and overcoming potential barriers to improved mastitis control. MASTITIS remains one of the most significant diseases affecting dairy cows in the UK and worldwide resulting in large economic losses to the industry and compromised welfare for the cows affected . DespiteA detailed mastitis control plan was devised and tested in a randomised controlled trial during 2004–2005 and found to reduce clinical mastitis (CM) and new infections as measured by somatic cell count (SCC) by approximately 20 per cent . It was A variety of studies have considered on-farm management practices relevant to mastitis control but there have been relatively few peer-reviewed studies from the UK  and nothFarms typically participated in the DMCP if they were concerned about udder health on their farm or their veterinarian had highlighted mastitis as an area for improvement as part of an ongoing herd health service. The plan would usually be delivered by the farm's own veterinary practice or by a dairy consultant. Veterinary practices could alternatively arrange for an external plan deliverer to implement the plan if they were not able to deliver it directly.The first stage of conducting the plan on the farm involved detailed data analysis whereby the mastitis epidemiology was described according to the likely mode of pathogen transmission involved  and the stage of lactation at which new infections were most often occurring as well as seasonality, parity and recurrence. A categorical herd ‘diagnosis’ was made based on this  and coulThe next stage involved a farm visit during which a comprehensive questionnaire was completed consisting of questions for the farmer and observations/measurements made by the plan deliverer. The questionnaire contained 377 questions in 12 sections consisting of general farm information, milking herd management between milkings, premilking management, milking routine, milking plant, postmilking management, management of the dry period, calving cows, treatment of mastitis, biosecurity, dairy replacements and mastitis monitoring. The answers to the questionnaire and the ‘diagnosis’ made were entered into a software package called the ‘DMCP ePlan’ . Once alThe plan deliverer would prioritise 5–10 of these interventions to be implemented on the farm. A three-level ranking system was used for the interventions based on the strength of evidence from research to assist the plan deliverer in prioritising which interventions to focus on; interventions supported by most evidence were made the priority for action .Participating farms were included in this study if the herd performance data (e.g. SCC data and CM records) were available at the plan start date in addition to the ePlan data .Herd performance data were submitted electronically by the plan deliverer when each farm was enrolled on the DMCP. Plan deliverers were contacted directly by the author and asked to send relevant ePlan data.The non-invasive nature of this project and anonymised data collection meant that formal ethical committee approval was not necessary.The herd performance and ePlan data were imported into Microsoft Access, checked and exported into Microsoft Excel for analysis. The herds were grouped accordingly for analysis: EDP, EL and CDP/CL. The CDP/CL herds were grouped together due to similarities in the epidemiology and low numbers of herds assigned those contagious diagnoses.The parameters used to measure mastitis performance in the participating herds are defined in The proportion of herds that were not performing each intervention was calculated and the frequency with which each intervention was ‘prioritised’ by the plan deliverers was also calculated. The interventions were ranked according to the proportion of eligible herds that undertook them and the interventions that were least commonly practised were reported .A total of 234 herds were included in the study that had been enrolled on the DMCP between 2009 and 2012. The geographical location of the farms is shown in Differences between the mastitis parameters for the different groups of herds are shown in The interventions that were most frequently found not to be undertaken in herds with different diagnoses are displayed in The three least commonly practised interventions in the EDP herds were the separation of heifers from dry cows before calving, allowing at least four weeks before returning dry cows to any one grazing, loafing or rest area after it has been used by cattle and not allowing dry cows to have access to any one lying area for more than two weeks. The three least commonly practised interventions in the EL herds were grouping cows with a high SCC/CM separately and milking them last at each milking, using hot disinfectant to clean clusters that become dirty during milking and milking cows with a high SCC/CM last.The results of this study show that many mastitis-related management practices that are generally considered to be important were not widely performed in a large sample of UK dairy herds. This is one of the most comprehensive field studies of its kind and the first to group the herds according to the putative origin of new mastitis cases. This grouping is important as the most significant aspects of mastitis control for a CL herd are very different from those for an EDP herd and therefore by grouping herds in this way, the authors are able to highlight the most relevant management ‘deficiencies’.While representing a relatively large sample of UK dairy herds for this type of study, it is likely that the results are biased towards herds seeking veterinary input with respect to mastitis control rather than being representative of the national herd as a whole. However, this may provide a true reflection of dairy herds seeking veterinary input with respect to mastitis control and therefore is of value to those involved in the delivery of these services. While reasonable steps were taken to ensure all data were accurate and complete, no formal validation of CM data was undertaken.Management of the dry cow/calving cow accommodation to maximise hygiene was an area of potential weakness highlighted in this study. Dry cows had continual access to the same pasture/lying area for more than two weeks in over 80 per cent of EDP herds and were allowed to return to paddocks within four weeks of them being previously grazed in 85 per cent of EDP herds. The ‘graze 2, rest 4’ strategy, that is, graze the same paddock for no more than two continual weeks followed by at least a 4-week rest period, has been found to be very effective at reducing the risk of CM in the first 30 days after calving  and was The size of the bedded lying area for dry cows was insufficient in over half of the EDP herds in this study despite research demonstrating the importance of this with respect to SCC in the first 30 days of lactation . Other pLess than 20 per cent of the EDP herds used individual calving pens despite evidence that they are associated with a reduced SCC and reduced incidence of CM . This inAlmost 60 per cent of the EDP herds were not selecting dry cow therapy (DCT) at cow level in this study (DCT products selected according to the infection status at drying-off) and this was made a priority in 45 per cent of the herds not doing so . Whole-hLess than 30 per cent of EDP herds were reducing yields to below 15 l before drying-off and this was only prioritised in 26 per cent of cases suggesting that other interventions were deemed more important for most EDP herds. Increased yields at drying-off have been associated with increased SCC  and IMI The vast majority (86 per cent) of EDP herds mixed the heifers with the cows before calving. However, several studies have demonstrated that the mixing of maiden heifers and cows during the dry period is associated with increased rates of CM  and incrFor herds with an EL diagnosis, key focus areas include the management of the milking cows’ environment as well as the milking routine and machine maintenance. The management of high SCC cows and those with CM featured prominently and it was interesting that ‘infected’ cows were rarely housed separately to the main herd in the present study despite good evidence of the benefits of doing so . Where tAnother aspect of management relating to the hygiene of the milking plant that was not widely practised was the replacement of liners at the appropriate interval. This highlights the value in the AHDB DMCP approach in that it ensures that mastitis control measures that are often assumed to be universally implemented are investigated and rectified when found to be lacking.The practice of foremilking was only carried out in approximately a quarter of the EL herds in this study. Foremilking is typically recommended to detect CM and is also a means of premilking stimulation . The appTwo-thirds of the EL herds were not following the ‘graze 2, rest 4’ principle as described previously and the same number of herds were allowing cows to wait for more than 1 hour to be milked. These aspects of environmental management could both result in an increased exposure of the cows teats to pathogens in addition to the increased risk of lameness caused by increased waiting times before milking .Many of the management practices least implemented by the CDP/CL herds were the same as for the EL herds and focused primarily on the risk of transmission during the milking process as would be expected. Due to the low number of herds with a CDP or CL diagnosis, they were combined as one group for the purposes of this study and the risk factors, therefore, were taken to be the same for both. Perhaps the most striking feature concerning these herds was how few of them grouped or milked cows according to their infection status or replaced the liners at the correct interval which for these herds is likely to be of paramount importance. This was reflected in the high proportion of such interventions that were prioritised by the plan deliverers for the CDP/CL herds.The majority of herds in this study (87 per cent) were classified as having a predominantly environmental pattern of disease divided almost equally between EDP and EL diagnoses. This was not unexpected as it reflects the national trend for the increased importance of the cows environment as a source of intramammary infections relative to the contagious spread of pathogens from cow to cow that was more common historically . ContagiThe importance of the dry period with respect to mastitis control has been well documented  and it iThe EDP herds had similar BMSCC and CM rates as the other herds in the study but were characterised by a significantly higher rate of CMDP than the other herds when the plan was first implemented as would be expected. They also had a significantly higher rate of DPNIR than the EL herds. Suggested targets for the rate of DPNIR and CMDP are 10 per cent and 1 in 12 (8.33 cases/100 cows/year), respectively, and the averages for the EDP herds in this study were considerably higher than these.Herds with an EL diagnosis had a similar BMSCC and CM rate to the other herds in the study but were characterised by significantly lower rates of DPNIR and significantly higher DPCURE rates than the other herds as well as a significantly higher rate of CML than the EDP herds. Suggested targets for LNIR and CML are 5 per cent and 2 in 12 (16.66 cases/100 cows/year), respectively.There were far fewer herds with a ‘contagious’ diagnosis in this study. The CDP/CL herds were characterised by a lower average milk yield than the other herds in the study and a higher BMSCC which would be expected due to the increased chronicity associated with IMIs caused by ‘contagious’ pathogens . All othThe frequency with which the interventions reported in this study were prioritised by the plan deliverers varied widely. When interventions were not highly prioritised, this may reflect the presence of more pressing concerns in those particular herds or perhaps a lack of perceived efficacy. It should be recognised that the process of prioritisation would be influenced by a combination of guidance from the ePlan and subjective assessment from the plan deliverer; it is uncertain the extent to which each of these elements influenced the final decisions made on prioritisation. With a limited number of intervention studies from which to draw from, it is very difficult to have much certainty about the efficacy of most mastitis interventions at the individual herd level and any uncertainty about the clinical and financial benefit of an intervention will affect the decision to implement it . A usefuThe results of this study provide data on performance and management of UK dairy herds, grouped according to the main putative origin of new cases of mastitis. Many aspects of management that might be considered to be important in mastitis control were not being practised by a large proportion of these herds. A better understanding of those practices not widely adopted by UK dairy farmers at present may aid practitioners in identifying and overcoming potential barriers to improved mastitis control in UK dairy herds.


The end-use quality of wheat flour varies as a result of the growth conditions of the plant. Among the wheat gluten proteins, the omega-5 gliadins have been identified as a major source of environmental variability, increasing in proportion in grain from plants that receive fertilizer or are subjected to high temperatures during grain development. The omega-5 gliadins also have been associated with the food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA). Recently, transgenic lines with reduced levels of omega-5 gliadins were developed using RNA interference (RNAi). These lines make it possible to determine whether changes in the levels of omega-5 gliadins in response to environmental conditions and agronomic inputs may be responsible for changes in flour end-use quality.Two transgenic wheat lines and a non-transgenic control were grown under a controlled temperature regimen with or without post-anthesis fertilizer and the protein composition of the resulting flour was analyzed by quantitative two-dimensional gel electrophoresis (2-DE). In one transgenic line, all 2-DE spots identified as omega-5 gliadins were substantially reduced without effects on other proteins. In the other transgenic line, the omega-5 gliadins were absent and there was a partial reduction in the levels of the omega-1,2 gliadins and the omega-1,2 chain-terminating gliadins as well as small changes in several other proteins. With the exception of the omega gliadins, the non-transgenic control and the transgenic plants showed similar responses to the fertilizer treatment. Protein contents of flour were determined by the fertilizer regimen and were similar in control and transgenic samples produced under each regimen while both mixing time and mixing tolerance were improved in flour from transgenic lines when plants received post-anthesis fertilizer.The data indicate that omega-5 gliadins have a negative effect on flour quality and suggest that changes in quality with the growth environment may be due in part to alterations in the levels of the omega gliadins. Because a known food allergen and one of the major sources of environmentally-induced variation in wheat flour protein composition has been eliminated, the transgenic lines may yield flour with both improved end-use quality and more consistent functionality when grown in different locations.The online version of this article (doi:10.1186/s12870-014-0393-1) contains supplementary material, which is available to authorized users. The growth conditions of the wheat crop cause changes in the protein composition of the grain as well as variations in the functional properties and allergenic potential of the resulting flour. However, it is difficult to establish links between changes in specific proteins and flour quality because of the complexity of the major wheat flour proteins. The main determinants of flour end-use quality, the gluten proteins, consist of gliadins and glutenins that together comprise about 70-80% of the flour protein. Both types of proteins contain repetitive sequences and an abundance of glutamine and proline and are largely insoluble in aqueous solutions ,2. The gEach of the gliadin and glutenin groups contains many similar proteins that also vary among wheat cultivars. Despite this complexity, a detailed map of the flour proteome in which most of the major flour proteins were linked to specific gene sequences was developed for the hard red spring wheat Butte 86 using two-dimensional gel electrophoresis (2-DE) combined with tandem mass spectrometry (MS/MS) ,8. TheseThe omega gliadins consist of two subgroups of proteins with distinct sequences, referred to as omega-5 and omega-1,2 gliadins. The omega-5 gliadins have N-terminal sequences beginning with SRL and the repetitive motifs FPQQQ and QQIPQQ. These proteins are of additional interest because of their involvement in the serious food allergy wheat-dependent exercise-induced anaphylaxis (WDEIA) ,11. In cRecently, RNA interference was used to silence the expression of genes encoding omega-5 gliadins in transgenic Butte 86 plants . Two traNon-transgenic Butte 86 and transgenic lines SA-8-35b-5 and SA-8-45a-2, referred to here as 35b and 45a, were grown in controlled temperature greenhouses with and without post-anthesis fertilizer (20:20:20 NPK) and average grain weights were determined at 7, 14, 21, 28, 35 and 42 days post-anthesis (DPA). Maximum fresh weights for all samples were achieved at 35 DPA. For plants grown without post-anthesis fertilizer, the maximum fresh weights ranged from 78 to 81 mg/grain. When plants were supplied with post-anthesis fertilizer, the maximum fresh weights were slightly greater and ranged from 85 to 93 mg/grain. Bushel weights of mature grain averaged 64.8 +/− 0.3 lb/bu when plants were grown without post-anthesis fertilizer and 63.2 +/− 0.4 with fertilizer. There were no significant differences between the bushel weights of grain from the control and transgenic lines under either regimen. However, there were notable differences in the appearances of mature kernels produced under the two regimens. As shown in Additional file Total protein was extracted from white flour milled from triplicate samples of the three lines produced under the two fertilizer regimens and analyzed by 2-DE in triplicate. Representative gels are shown in Figure Volumes of individual 2-DE spots determined from each of the technical replicate gels were averaged and the average spot volumes were then determined for the biological replicates  were sent to the Hard Winter Wheat Quality Laboratory (HWWQL) and milled to straight-grade flour using methods standardized by the American Association of Cereal Chemists (AACC Approved Methods 26–10.02 and 26–22.01) [Total protein was extracted from 50 mg samples of flour from each of the biological replicates with SDS under reducing conditions as described previously . ProteinGels were digitized using a calibrated scanner at 310 dpi . 2-DE spots were matched between gels, quantified and normalized using SameSpots v. 4.5 . Five hundred ten spots were detected and quantified by the SameSpots software.Comparisons were made between triplicate gels from each of the three biological replicates for non-transgenic and transgenic lines 35b and 45a under each growth regimen using the SameSpots software. Comparisons also were made between the two growth regimens for each line. Differentially expressed spots were identified using analysis of variance (ANOVA) performed by the SameSpots software. All differentially expressed spots had ANOVA values <0.01 and showed at least 1.2-fold changes.Of the 510 spots analyzed, 220 of the most abundant protein spots had been identified previously from Butte 86 flour by MS/MS . The aveAverage volumes of all spots identified as either HMW-GS or LMW-GS  were summed for each flour sample and used to determine the ratio of HMW-GS to LMW-GS in that sample. Likewise, average volumes of all spots identified as gliadin  or glutenin (HMW-GS and LMW-GS) were summed and used to determine the ratio of gliadin to glutenin in that sample. Spots grouped in each gluten protein family are shown in Additional file End-use functionality tests  were conducted on each of the biological replicates at the HWWQL  using American Association of Cereal Chemists (AACC) approved methods that are routinely used for end-use quality assessment of breeding lines . ProteinAll supporting data for this manuscript are included in Additional files.


ATRX gene encodes hATRX, a chromatin-remodeling protein harboring an helicase/ATPase and ADD domains. The ADD domain has two zinc fingers that bind to histone tails and mediate hATRX binding to chromatin. dAtrx, the putative ATRX homolog in Drosophila melanogaster, has a conserved helicase/ATPase domain but lacks the ADD domain. A bioinformatic search of the Drosophila genome using the human ADD sequence allowed us to identify the CG8290 annotated gene, which encodes three ADD harboring- isoforms generated by alternative splicing. This Drosophila ADD domain is highly similar in structure and in the amino acids which mediate the histone tail contacts to the ADD domain of hATRX as shown by 3D modeling. Very recently the CG8290 annotated gene has been named dadd1. We show through pull-down and CoIP assays that the products of the dadd1 gene interact physically with dAtrxL and HP1a and all of them mainly co-localize in the chromocenter, although euchromatic localization can also be observed through the chromosome arms. We confirm through ChIP analyses that these proteins are present in vivo in the same heterochromatic regions. The three isoforms are expressed throughout development. Flies carrying transheterozygous combinations of the dadd1 and atrx alleles are semi-viable and have different phenotypes including the appearance of melanotic masses. Interestingly, the dAdd1-b and c isoforms have extra domains, such as MADF, which suggest newly acquired functions of these proteins. These results strongly support that, in Drosophila, the atrx gene diverged and that the dadd1-encoded proteins participate with dAtrx in some cellular functions such as heterochromatin maintenance.The human  ATRX gene (hATRX) was described approximately 20 years ago as the main gene mutated in ATRX syndrome . ATRX is localized on the X chromosome in position Xq13.1–q21.1. Individuals with mutations in this gene present several phenotypic charateristics that may include mental retardation, craniofacial and urogenital deformities, psychomotor failure and alpha-thalassemia ATRX, DNMT3b and DNMT3L). It was recently demonstrated through different in vitro and in vivo approaches that this domain recognizes the combination of K9me3 and unmethylated K4 residues of the histone H3 tail in vitro ATPase activity, which can be stimulated both by DNA and nucleosomes The human Genome-wide studies have identified hATRX as a protein that is able to bind to DNA regions that can acquire a G4 structure conformation, such as telomeres and repetitive G-rich regions ATRX gene is highly conserved through eukaryotic evolution, but in invertebrates and particularly in Drosophila, it encodes proteins that lack the ADD N-terminal domain, i.e., the dAtrxL and dAtrxs isoforms are encoded by the same gene but lack an ADD domain. The dAtrxL isoform interacts with HP1a and is localized to heterochromatin, whereas the dAtrxs isoform is localized to euchromatin It is clear though that both the ADD and helicase/ATPase domains play crucial roles during development Drosophila that could encode proteins with the conserved the amino-terminus of the vertebrate ATRX. Performing in silico analyses, we demonstrate that the annotated gene CG8290, recently named dadd1L. Using a genetic approach, we found that these proteins have important functions during Drosophila development and that they cooperate with dAtrxL in certain functions. The evidence leads us to propose that the dAdd1 proteins are orthologs of the amino-terminus of the ATRX protein in vertebrates.We decided to determine if there was a gene in 2 euthanasia.All animal handling was approved by the Instituto de Biotecnología Bioethics Comittee, Permit Number 344 (2011/02/10), which follows NOM-062 animal welfare mexican law. All efforts were made to minimize animal suffering. Animals were sacrificed by COThe domain organization of dAdd1 isoforms  and the Multiple alignments were performed with CLUSTALX2 2.1 Homo sapiensIn an attempt to determine a protein-modeling of ADD domain (101 amino acid residues), we generated by SWISS-MODEL OreR) or 1118w, and fly stocks were maintained at 25°C with standard food. The stocks that carried the atrx alleles were obtained from the Bloomington Stock Center. The stocks that carried dadd1 and Su(var)2-5 alleles were obtained from the Drosophila Genetic Resource Center (DGRC), Kyoto Institute of Technology. The xnp/atrx alleles  were described by Bassett AR, et al., (2008) dadd1 alleles: NP0793dadd1  and NP1240dadd1 , carry a P-element insertion at -225 and -223 bp from the translation initiation codon of the dadd1 gene, respectively. The 2Su(var)2-5 allele is a missense mutation that has been characterized molecularly by Eissenberg et al., (1992) 5Su(var)2-5 allele is an X-ray induced mutation, in which only the first 10 amino acids of HP1a are translated hsp70-lacZ transgenic reporter, with the reporter gene positioned adjacent to a 3L pericentric heterochromatin mass The wild-type flies used in this study were Oregon R  larvae. The Scer\UAS.T:Ivir\HA1Xnp allele is described in Sgs3-GAL4  expression to salivary glands from third instar larvae. Both latter lines were obtained from the Bloomington Drosophila Stock Center.Polytene chromosomes for immunostaining were obtained from wild type L antibody at 1∶100, pan-dAdd1 at 1∶50 and anti-HA (Roche) at 1∶50. Secondary antibodies were Alexa fluor 488 used at 1∶500 and Alexa fluor 568 or 594 used at 1∶100 (Invitrogen). Images were taken on a confocal laser-scanning microscope (Olympus FV1000) at the Laboratoratorio Nacional de Microscopía Avanzada .Immunostaining of polytene chromosomes was performed with slight modifications of the protocol described in L (dAtrxL-HA) because the dAdd1 and dAtrxL antibodies were raised in the same species and could not be used together for this experiment.For the double dAdd1/dAtrx immunostaining we followed an epitope tagged version of dAtrxL and the dAdd1 proteins. All the clones used in this work were nucleotide-sequenced. The Drosophila LD28477, LD24316 and LD37351 cDNA (BDGP Gold collection of Drosophila Genomics Resource Center) were amplified by PCR and cloned in the EcoRI, NotI, SmaI, XhoI or SalI sites of the pGEX-4T1 or pGBKT7 vector (details are available upon request). The dAtrxL and dAdd1 fragments were expressed as GST fusion proteins in a bacterial system. For interaction assays, over-expression of dAtrxL fragments fused to GST in bacteria was induced with 0.4 mM IPTG during 3 h. GST-dAtrxL fragments were purified using glutathione-sepharose (Amersham) according to manufacturer's instructions. The dAdd1 fragments were expressed and labelled with S35 using an in vitro transcription/translation system (TNT-Quick-Coupled Transcription/Translation System from Promega). Pull-down assays were performed according to the manufacturer's instructions.We generated fusion proteins of several fragments of dAtrxEcoRI/NotI sites of pAc5.1/V5-HisA vector (Invitrogen). Drosophila S2R+ cells were maintained in Schneider medium with 10% fetal bovine serum and 100 µg streptomycin/0.25 µg amphotericin. Cells were cotransfected with 10 µg of each construction by the calcium method (Invitrogen). Fourty-eight hours after transfection, the cells were collected and lysed. Lysates were clarified by centrifugation at 15.7 g at 4°C. CoIPs were performed as indicated in The dAdd1-a cDNA was cloned into st, 2nd and 3rd instar larvae , pupae (P), pharate (Ph) and female and male adults Oregon R flies (F and M). 10 µg of total RNA was converted to cDNA using reverse transcriptase enzyme (Invitrogen) and oligo-dT and random primers (Stratagene®). To assess the presence of the transcripts the following oligonucleotides were used for the PCR reaction: dAdd1-a forward (5′CATCTTACGGGCAAAGTGGT-3′); dAdd1-a reverse (5′CAGGCTGGCCAATATCGTGG-3′); dAdd1-b forward (5′GCTTGTCATCGGGCATATCT-3′); dAdd1-b reverse (5′GCTCATAAGCAGCCAGCAGT-3′); dAdd1-c forward (5′ACAGCGGCAGCAACGGAAGC-3′); dAdd1-c reverse (5′GCGGAAGTCCTTGCAGCGGT-3′); rp49 forward (5′TCAAGATGACCATCCGCCCA-3′); rp49 reverse (5′GTTCTCTTGAGAACGCAGGC-3′). rp49 was used as an RT- PCR control.RNA was obtained using the Trizol reagent (Invitrogen®) from embryos (0–3 and 3–21 hour post-fertilization), 15′-CAACCAAGACCAACCTACCC-3′); rover reverse (5′-GCTCATTTTAGTCTGTCCGC-3′) 5′-TGACTGCCTCTCATTCTGTC-3′) and TAS3L_ChIP2 (5′-TATCATCTCGTTCATCCGCC-3′) S2R+ cells or third instar larvae salivary glands from wild type organisms were fixed in 1% formaldehyde. The fixation reaction was stopped by adding glycine (125 mM). Cells or salivary glands were washed and resuspended in lysis buffer, and sonication was performed until the size of chromatin reached between 200 and 800 bp. Pre-clearing, antibody incubations, immunoprecipitation and phenol:chlorophorm extractions were performed as described in Detection of β-galactosidase in adult flies was performed as in Gu and Elgin (2013) Drosophila genome using the ADD region (aa 169-268) of the hATRX protein with the BLAST  of the NCBI . Using this approach, we identified the CG8290 annotated gene, which putatively encodes three annotated proteins that have a conserved amino-terminal ADD domain that is 52% homologous and 36% identical to the hATRX ADD domain (Drosophila CG8290 encoded proteins is more similar to the one in hATRX than in DNMT3L (data not shown). While this work was in preparation for publication Alekseyenko et al., (2014) named the CG8290 gene dadd1We searched for genes encoding proteins with the ADD domain in the D domain . ProteinD domain  showed tDrosophila and in hATRX (aa 110-126 in dAdd1) Drosophila domains mainly overlap in the GATA-like and PHD zinc fingers helices 1, 2 and 3 , and the pocket that is important for recognition of H3K9me3 and H3K4me0 is also conserved , the capability of this domain to bind to the H3K9me3 tail was assayed, confirming it preferentially binds to this histone modification In the hATRX protein, there are some amino acids in a "hydrophobic pocket" that mediate the interaction between the H3K9me3 and unmethylated H3K4 (H3K4me0) histone tail combination. In general, it is noteworthy that the whole "hydrophobic pocket" binding site is conserved between the ADD in onserved . In the monkey king (mkg) gene family in Drosophila) We also compared the native structure of the ADD domain of dAdd1 to the ADD domain of DNMT3L (data not shown) and found less overlaping when we superimpose the structures. This led us to propose that during ATRX evolution, the protein may have undergone a fission event. To obtain insight into the evolutionary history of ATRX, we decided to perform a rooted phylogenetic inference analysis using homologous sequences of the ADD and the helicase/ATPase domains from ATRX of higher eukaryotes. We found that the shared common ancestor of higher eukaryotes possessed a protein with both the ADD and helicase/ATPase domains, but in insects, it underwent a fission event by which the two domains were separated, generating two different genes , Fig. S2Drosophila, ADD harboring-proteins acquired other domains such as MADF , suggesting novel functions for these proteins  corresponded to the ones derived from alternatively spliced transcripts of CG8290 described in FlyBase Through RT-PCR analyses of cDNAs obtained from different developmental stages see , we foundAdd1-b transcript, another higher molecular weight band appears (Interestingly when we amplify the We found that there are three proteins derived from these transcripts that have similar predicted molecular weights ranging from 112 kDa to 130 kDa . Interes+ cells (We found that the dAdd1 proteins are enriched in the nuclear fraction of S2R+ cells . Because+ cells . The sig+ cells .L (dAtrxL-HA) L-HA version localizes to the same sites as wild type dAtrxLL-HA in the chromocenter and in other heterochromatic regions , the specific bands obtained in the IP are marked with a white arrow. We also performed the reciprocal immunoprecipitation with the dAtrxL antibody, and we show that at least one of the dAdd1 proteins, approximately 130 kDa, co-immunoprecipitates with dAtrxL  (To map the dAdd1 protein domain(s) important for the interaction with dAtrxstem see , Fig. 3CL fragments (1-221 and 368-1103), corresponding to the amino-terminal and SNF2 fragments, whereas the second dAdd1-a fragment (620-1199), which lacks amino acids 355-625, fails to interact with any of the tested dAtrxL fragments  and dAdd1-a (620-1199) fragments. The first fragment, which contains amino acids 355-625 binds to two dAtrxL protein  interacts with the amino terminal and SNF2-containing fragments of the dAtrxL is conserved in all dAdd1 isoforms. Thus, all dAdd1 isoforms can directly interact with the amino-terminal domain of dAtrxL (a.a. 1-221) through their amino-terminal domain (a.a. 1-625). Interestingly, this dAtrxL fragment is only conserved in the long isoform, and it does not overlap with the HP1a interaction domain L protein.From these data, we conclude that the dAdd1 domain that interacts with dAtrxatrx and dadd1 flies. For these analyses, we used three atrx alleles 1atrx is a deficiency that uncovers dAtrx as well as three more adjacent genes; 2atrx is an hypomorphic allele that affects both the long and short dAtrx isoforms and 3atrx is an hypomorphic allele that affects only the long isoform. The survival of heteroallelic atrx flies is affected, as previously reported, and they do not present any other obvious phenotype  and thus we used for the rest of our tests the NP1240dadd1 allele. 44% and 90% of the NP1240/dadd1NP1240dadd1 and NP1240/dadd1NP0793dadd1 flies reach adulthood respectively, showing that the higher lethality present in NP1240/dadd1NP1240dadd1 individuals (56%) compared to the one presented by the NP1240/dadd1NP0793dadd1 flies (only 10%) may be caused by other lethals present in the NP1240dadd1 chromosome  assay of the V5-dAdd1 protein with the V5 antibody  assays using different antibodies against HP1a, dAtrxells see .et al., (2012) CG8290 are enriched in the telomeric associated sequences (TAS-L) but they did not find HP1a. Thus, we looked for the presence of HP1α, dAtrxL and the dAdd1 proteins in the telomeric region (TAS-L). Our ChIP experiments show, similar to Antão et al., (2012) L and the proteins, but not HP1α, are enriched in the TAS-L region  (HP1α is not enriched in the TAS (Telomeric Associated Sequences) regions L region . We alsoure (G4) .L is present but the dAdd1 proteins are not enriched in this region. This result indicates that dAdd1 and dAtrxL do not colocalize at all the heterochromatic regions in the genome  The colocalization of HP1a, dAtrxL localize at the chromocenter of polytene chromosomes and act as suppressors of position effect variegation for the m4w allele, an inversion that lies near pericentric heterochromatin It has not been determined conclusively whether Drosophila dAtrx is involved in the deposition of the histone variant H3.3, but both HP1a and dAtrxdadd1 capability to suppress position effect variegation. We used the BL1 line, which carries a LacZ reporter construct that lies near the centromere dadd1 is involved in the establishment, crossing virgin females carrying the dadd1 alleles to males carrying the reporter BL1 , or maintenance, crossing males carrying the dadd1 alleles to females carrying the reporter BL1 (zygotic effect), of heterochromatin Following this hypothesis we tested the dadd1 does not have a relevant role in the establishment of pericentric heterochromatin  bar and C (control) bar in dadd1 male carrying alleles, the activity of LacZ was consistently higher in dadd1 mutants (compare Z (zygotic) bar to C (control) bar in dadd1 alleles can suppress position effect variegation and that dadd1 has a role in the maintenance of heterochromatin rather than in the establishment of heterochromatin.The results show that dadd1 and atrx alleles enhances the suppression of position effect variegation in a cooperative manner. In fact, this is what we observed. Our results show that transheterozygous dadd1/atrx flies suppress position effect variegation more efficiently than heteroallelic flies carrying either the dadd1 or atrx alleles 205 is a loss-of-function HP1a allele because it carries a mutation in aminoacid 26 within the chromodomain. We tested whether the loss of function of HP1a in the presence of dadd1 and/or atrx mutations affects the presence of the melanotic masses found in dadd1/atrx individuals.We showed that 2Su(var)205 flies carrying the dadd1, or 1atrx or 2atrx alleles do not present melanotic masses, whereas 2Su(var)205 flies carrying 3atrx (which affects only the long isoform of dAtrx) present melanotic masses in a small percentage  or 2Su(var)205/+; 3atrx/+ (2%) individuals increases to 14% in 2Su(var)205/NP1240dadd;3atrx/+ individuals where mutations in the three genes are together 205 flies most likely involves misregulation in the maintenance of heterochromatin.Transheterozygous rcentage . The pentogether . These dDrosophila dAdd1 proteins as orthologs to the amino-terminal region of Atrx. The dadd1 gene expresses the dAdd1 proteins, which have an ADD domain , which is the homolog of the amino-terminal portions of mammalian MLL2 and MLL3Our phylogenetic inference analysis using the ADD domain of hATRX indicates that the ATRX gene underwent a fission event. This fission has been described in at least another chromatin-binding-protein encoding gene, dadd1 gene. The dAdd1-a isoform contains the ADD domain and no other putative domains, while the other two spliced isoforms have additional domains called MADF (myb/SANT-like domain in Adf1). The MADF domains are a subfamily of the SANT domains. They are DNA-binding domains found in all Drosophila species. In the case of Adf1, the first Drosophila factor identified to have an homology to the Myb oncoprotein It is intriguing that there are three spliced isoforms encoded by the The predicted isoelectrical point of the three MADF domains in the dAdd1 proteins is basic, which could indicate that they can bind to DNA.L. This indicates that the dAdd1 isoforms could also have roles independent of their interaction with dAtrxL.The acquisition of new domains indicates that these proteins most likely have diverged functions. One indication of this could be the pattern of the immunolocalization of dAdd1 in polytene chromosomes, where not all the signals derived from the pan-dAdd1 antibody co-localize with dAtrxdadd1/atrx alleles is compromised, and some of the larvae and adult individuals show melanotic masses. We found that these proteins also colocalize with HP1a and that dAdd1-a immunoprecipitates with HP1a. Diminished HP1a levels in dadd1/atrx flies enhance the incidence of melanotic masses. Our results are supported by the recent report of Alekseyenko et al., (2014), in which it is demonstrated that dAdd1 physically interacts with HP1a and that dAdd1 is a suppressor of variegation The survival of transheterozygous individuals carrying the pannier (pnr) as a gene regulated by dAtrxL and another transcriptional factor DREF L is recruited to the pnr gene promoter through DREF, and that it acts as a co-repressor of pnr gene expression.The generation of melanotic masses involves problems in the differentiation of hematopoietic cells in the flies. The differentiation of hematopoietic cells takes place in the early embryonic head mesoderm and in the lymph gland of late larvae, and three different types of cells are derived from the prohemocytes; the plasmatocytes, crystal cells and lamellocytes. Several different pathways regulate the differentiation of these cells dadd1, atrx and 2Su(var)205 alleles indicates that melanotic masses are derived from problems in the proteins involved in heterochromatin maintenance, such as dAtrxL and HP1a. The position effect variegation assay indicated that the dAdd1 proteins are involved in the maintenance of heterochromatin. Thus, an attractive hypothesis is that dAdd1, dAtrxL and HP1a cooperate and promote heterochromatinization at the promoters of the genes involved in the JAK/STAT pathway, including pnr. Lack of these proteins could lead to misregulation of the effectors of the pathway, giving rise to the melanotic masses. Experiments are being carried out to test this hypothesis.Furthermore, the data obtained from the genetic interactions between the In the human ATRX syndrome, the majority of the mutations identified so far affect the ADD or the SNF2 helicase/ATPAse domains. The fact that the domains are separated in flies provides a new important tool to study the individual roles these domains have in the development of the organism.Figure S1dAdd1 antibodies recognize specifically the dAdd1 proteins. a) Western blot using the pan-dAdd1 antibody. The dAdd1 isoform signals (lane 1) observed with the pan-dAdd1 antibody are no longer observed in lane 2. This demonstrates that the GST-dAdd1 fusion protein which harbors the peptide used to raise the pan-dAdd1 antibody is able to deplete them from this fraction. The dAdd1 signals are no longer observed (lane 2) showing that the pand-dAdd1 specifically recognizes the dAdd1 isoforms  was performed using the anti-dAdd1-b antibody. A specific signal is observed  which corresponds to the induced GST-dAdd1-b fragment (aa 530-1125) and not GST or GST-dAdd1-a (aa 620-1199) (lanes 1 and 2 respectively). Faint bands in the first two lanes are unspecific signals.see also . b) Anti(TIF)Click here for additional data file.Figure S2The atrx gene suffered a fission event in the Insecta class. Maximum Likelihood Phylogenetic Analysis of Helicase/ATPase domain and the corresponding Protein Domain Architecture information of its containing proteins. The numbers shown represent bootstrap values. It can be seen that the common ancestor to plants and animals had a protein with both, the ADD and the helicase/ATPase domains, but insects show the homologous domains in different proteins. Since it is more likely that only one fusion event, instead of two independent acquisitions of the same domain, occurred during the evolution, the most parsimonious explanation is to consider a model in which a gene fission event occurred within the Insecta class. For the parameters used, see (DOCX)Click here for additional data file.Figure S3The dAdd1 proteins localize at many chromatin regions in polytene chromosomes. Wild type polytene chromosome staining was performed with the pan-dAdd1 antibody (red). The dAdd1 proteins localize in heterochromatic regions such as the chromocenter and the fourth chromosome . Staining along the chromosomes arms and in euchromatic regions is also observed.(TIF)Click here for additional data file.Figure S4Alleles NP1240dadd1 and NP0793dadd1 are hypomorphs. Semiquantitative RT-PCR from wild type and mutant dadd1 flies. dadd1 mRNA level is lower in the NP1240dadd1 (upper panel) and NP0793dadd1 (lower panel) heterozygous flies than in the dadd1 wild type flies (1118w). In homozygous NP1240/dadd1NP1240dadd1 (upper panel), dadd1 is even lower that in the heterozygous condition. rp49 transcript levels remained unchanged in the mutant alleles.(DOCX)Click here for additional data file.Figure S5The dAdd1 proteins co-localize in vivoL and HP1a in some chromatin regions. with dAtrx ChIP assay using total extracts from third instar salivary glands (SG) prepared from wild-type larvae and S2R+ cells. Graphs represent the percentage of input precipitated using the different antibodies for the same regions. Note that in the rover region only dAtrxL is enriched. Three independent biological replicates were performed.(TIF)Click here for additional data file.


A two-step process of phase determination in the X-ray structural analysis of the coat protein of a betanodavirus is described. A new indicator, the free fraction, for molecular averaging in real space is introduced to effectively evaluate the phasing power in order to enhance the success of determining new structures. ab initio phase determination and phase improvement. Applications of the cross-crystal averaging (CCA) method have been shown to be effective for phase improvement after initial phasing by molecular replacement, isomorphous replacement, anomalous dispersion or combinations of these methods. Here, a two-step process for phase determination in the X-ray structural analysis of a new coat protein from a betanodavirus, Grouper nervous necrosis virus, is described in detail. The first step is ab initio structure determination of the T = 3 icosahedral virus-like particle using NCS averaging (NCSA). The second step involves structure determination of the protrusion domain of the viral molecule using cross-crystal averaging. In this method, molecular averaging and solvent flattening constrain the electron density in real space. To quantify these constraints, a new, simple and general indicator, free fraction (ff), is introduced, where ff is defined as the ratio of the volume of the electron density that is freely changed to the total volume of the crystal unit cell. This indicator is useful and effective to evaluate the strengths of both NCSA and CCA. Under the condition that a mask (envelope) covers the target molecule well, an ff value of less than 0.1, as a new rule of thumb, gives sufficient phasing power for the successful construction of new structures.Molecular averaging, including noncrystallographic symmetry (NCS) averaging, is a powerful method for  The feasibility of ab initio phase determination has been demonstrated for spherical viruses  averaging and cross-crystal averaging (CCA), coupled with solvent flattening, has proved to be powerful in improving the determination of the phases in protein crystallography. As a case in point, NCS averaging (NCSA) with phase extension of icosahedral viruses is a common procedure for phase improvement after initial calculations based on molecular replacement (MR) using a density map from a cryo-electron microscope, a similar model or initial experimental phases from isomorphous replacement or anomalous dispersion. Since the successful structure determination of the human common cold virus serotype 14 , have been solved. Various strains of the genus Betanodavirus have been isolated from several fishes, such as Striped jack nervous necrosis virus (SJNNV), Tiger puffer nervous virus (TPNNV), Red-spotted grouper nervous necrosis virus (RGNNV) and Barfin flounder nervous necrosis virus (BFNNV) , in various forms: a complete T = 3 GNNV-like particle (GNNV-LP) at 3.6 Å resolution, a T = 1 icosahedral subviral particle of the truncated surface domain (S-domain) at 3.1 Å resolution and the individual protrusion domain (P-domain) of the GNNV coat protein (CP) at 1.2 Å resolution  of T = 3 GNNV was successfully obtained. Atomic modelling with the amino-acid sequence of the S-domain of the T = 3 GNNV capsid protein was straightforward. The density map of the P-domain region in the T = 3 GNNV-LP was, however, insufficient for complete model building, so that only partial main chains could be traced. An analysis of the T = 3 GNNV particle structure led us to design a truncated form consisting of only the P-domain of the CP. Several crystal forms of the individual P-domain were subsequently obtained at high resolution . Secondly, utilizing the cut-out envelope of the P-domain region of the T = 3 GNNV-LP crystal, we applied CCA to the various crystal forms of the P-domain to acquire the correct phases, including the phases for the high-resolution data. In this manner, the structure of the individual P-domain was successfully built with good quality and was subsequently used for complete model building and refinement of T = 3 GNNV-LP.To determine the new structure of GNNV-LP, we applied molecular-averaging methods in two steps. Firstly, an NCSA Following earlier work  detector . The crystals of GNNV-LP belonged to the monoclinic space group C2, with unit-cell parameters a = 477, b = 422, c = 337 Å, β = 134°. Diffraction images were collected with an oscillation angle of 0.3° per frame and an exposure duration of 3 s at a wavelength of 0.9 Å. The high-resolution data set (50–3.6 Å) was collected first.Crystals of ab initio phase determination, data at very low resolution are essential. During data collection, the beamstop was moved to the downstream side at 95 mm from the crystal, as near to the detector as possible; an X-ray beam of wavelength 1.2 Å was used to include the lowest resolution spots (266–12 Å). The images at this low resolution were collected with an oscillation angle of 0.5° per frame and an exposure duration of 3 s. The entire data sets, including those at very low and high resolutions, were recorded with an X-ray beam size of 50 µm on separate diffraction regions of a long GNNV-LP crystal of dimensions 0.1 × 0.1 × 0.3 mm.To assist HKL-2000  = (3 1 −1) and (3 1 −2), were missed in the resolution range below 100 Å. A total of 600 frames were taken for the high-resolution data set and 360 frames for the low-resolution data set; scaling was performed concurrently with these 960 frames in total. The data set at the highest resolution eventually contained 499 184 reflections from 50 to 3.6 Å with a completeness of 98% for use in refinement.For data processing, including integration and scaling, we used 2.2.MOLREP  =  from the icosahedral convention of orthogonal coordinate axes and fold axes: 2(z)-5-3-(x)2 .Self-rotation functions were calculated to determine the orientations of the icosahedral symmetries  using ab initio phasing method, which used NCSA with phase extension, an appropriate mask was necessary to separate two regions: the protein region to be NCS-averaged and the solvent region to be flattened. The initial mask for NCSA and solvent flattening was created from the atomic structure of T = 3 FHV (PDB entry 4fsj), a disparate structure, with a larger mask radius of 11–13 Å around each atom  and inverse FT (FT−1). In this cycle, NCSA and solvent flattening were applied to the density map in real space. A phase combination of Φcalc with w × Fobs by Rayment weighting  to calculate the weighting factor. RSTATS (coded by S. E. V. Phillips) was used to scale Fcalc to Fobs and to calculate R factors and correlation coefficients between Fobs and Fcalc.Using data with the ‘initial’ parameters of the above-described initial mask, the initial spherical model and the initial NCSA matrices, a; a is the unit-cell dimension). This process is referred to as ‘the procedure’ hereafter. The numbers of both iterations (>100 cycles) at low resolution and phase-extension steps (>50 cycles) sufficed for convergence  with 50 iterations in one reciprocal-lattice step .The electron density of helical structural elements such as α-helices in GNNV-LP at 3.7 Å resolution (or better) was used to examine the correct enantiomorphism of the phase sets Fig. 5. To impr2.3.DM  PEG 4000 (C2), 0.2 M KCl, 0.1 M magnesium acetate, 0.05 M sodium cacodylate pH 6.5, 10%(w/v) PEG 8000 (P212121) and 0.2 M NaCl, 0.1 M Tris–HCl pH 8.0, 20%(w/v) PEG 4000 (P3). All crystals appeared within one week. Single crystals (∼0.1 × 0.1 × 0.1 mm) were transferred to the respective crystallization solution containing glycerol (20%) as a cryoprotectant and were flash-cooled with liquid N2 for data collection.From the information of the corresponding sequence of the P-domain of 3.2.HKL-2000 . All data sets were processed with s Table 1. From ans Table 1, C2D was3.3.An initial attempt to determine the structure of the P-domain using the MR method with the previous partially traced model of the P-domain from GNNV-LP was not straightforward and failed. We then performed the CCA method using the three data sets P212121, C2D and C2S. A flow chart showing the contribution of each crystal form of the P-domain to phasing calculations is shown in Fig. 6T = 3 GNNV-LP, the coordinates of the  grid points of the map in the region of the P-domain with density greater than 0.4σ were selected. On these grid coordinates (not on density peaks), dummy C atoms were placed to form an envelope of the P-domain as a PDB-format file. This P-domain envelope served as the initial MR model for each crystal. Using Phaser . The correlation-coefficient values were 0.617, 0.779 and 0.710 for P212121, C2D and C2S, respectively. After phase improvement by CCA, the C2S data were used to build the model, because its resolution was the highest (1.80 Å) at the time. 4.4.1.ab initio method with NCSA and phase extension has been successfully implemented to determine the structure of the T = 3 GNNV icosahedral VLP. The key to success was the similarity of the mask (or the envelope) to the target structure. Despite significant variations in the main chains of the CP between FHV and GNNV, the envelopes of these T = 3 capsids are somewhat similar to each other, especially in the spatial position of the P-domain at the quasi-threefold axis. Considering that the P-domain of FHV is much smaller than that of GNNV, the radius of the mask was typically increased to 13 Å to cover the entire structure of GNNV. Other trials using the large spherical-shell mask, which was cut by the bisector planes between neighbouring virus particles, were not successful in phasing. On inspection of this spherical-shell mask, we found that it was so small, covering only half of the P-domain, as to result in a negative effect on the phasing. Tests investigating the simulation trials using errorless amplitudes, Fsimulated, which were calculated from the FHV model positioned in the unit cell of the T = 3 GNNV-LP crystals, led to a map with a rough envelope that was uninterpretable in the first trial. In the second trial using a revised mask, which was made from the rough envelope, an interpretable map was obtained even with the mask cutting a small portion of the P-domain. This result is similar to that described in a previous report . ff is a simple indicator, which is easily calculated, while general to NCSA and CCA. When the copy number of NCS is 1, the ff value is identical to a fractional volume of protein molecules to be determined in the cell volume.n, ff is expressed asS is the fraction of the solvent region. The solvent region is constrained to a constant value with solvent flattening. Furthermore, by the averaging restriction, the electron density of 1/n of the protein region can only be changed to reproduce the amplitude Fobs without constraint. Alternatively, it can be explained as that the densities of the 1 − 1/n protein fractional volume are restricted to the same densities as that of the 1/n protein volume.When the copy number of NCS is kS is the fraction of the solvent region of the kth crystal, kP is the protein fraction of the kth crystal and the equation Sk + Pk = 1 holds. A detailed derivation of (3)ASk + Pk ≠ 1, for example where the solvent mask and the NCS mask are different; kP can be substituted by 1 − kS.We can thus expand the same concept for the CCA case,m pieces of crystal, the kth crystal has kn NCS. We can writeIf we generalize the equation to BThis ff value is another expression for the ratio of the number of data to the number of parameters  for averaging was 30. The ff value was hence 0.024 (= 0.71/30). We performed trial calculations varying the ff values by decreasing the copy number of NCS with the initial FHV mask and with the initial NCS matrix obtained from the results of self-rotation functions. The results are summarized in Table 2When R factor and CC values have a tendency to deteriorate with an increased number of NCSA or a decreased ff value because of the decreased number of degrees of freedom. Considering that the superior R factor and CC values are not invariably meant to help to obtain the correct phases, it is thus necessary to take these common phenomena into account in parameter searching when monitoring criteria such as R factor and CC. The phase differences from the refined model are presented in Table 2The quality of the map deteriorated with a smaller copy number of NCS, as expected. Notably, the ab initio phasing.We also performed trial calculations varying the ff values by altering the mask volume. To increase or to decrease the mask volume, we extended or decreased the inner radius of the mask of FHV while the outer particle side of the mask was fixed. The mask with the inner radius extended to either 110 or 90 Å resulted in a non-interpretable density map. The trend in the variation of the ff value is difficult to obtain from simply varying the sizes of the mask volume. Not only a good coverage of the target molecule but also a suitable shape of the mask or envelope is critical for the success of ARP/wARP, compared with those in the lower rows, which failed. The trimeric symmetries were included in the calculation of the successful upper case, but were excluded in the calculation of the failed lower case; the same mask was used in both calculations. The R factor and CC values seem to be random, and might be affected by the structural similarity of each molecule and the validities of their transfer matrices, although these values deviate less in the upper case. In both cases, phase extension was not utilized because of a lack of low-resolution (>30 Å) data. The phase difference (57.4°) from the refined model was smaller in the successful case, whereas the value of the phase difference (81.8°) for the failed case was large and did not decrease from the initial value. ff seems to act as an effective indicator in these phasing calculations.The phasing results of the truncated P-domain with various ff values are summarized in Table 3The other factors in molecular averaging, such as the mask, transfer matrices and molecular equalities, as well as the data quality, must also be considered and evaluated. In the NCSA case, the mask roughly resembled the target envelope in shape and completely covered it. The transfer matrices and molecular isomorphism were expected to be satisfactory in the surface domains because of the nature of the icosahedral symmetry. The data quality, including the completeness of the very low resolution data, was essentially satisfactory. An ff value of ∼0.1 seems to be appropriate to separate successful (<0.1) and unsuccessful (>0.1) calculations in terms of map qualities for model building with NCSA Table 2. In the 10.1107/S2059798316007695/mh5197sup1.pdfSupplementary Figure S1.. DOI: