Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme of Lactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.The cell envelope proteinase (CEP) of  Bacillus protease subtilisin (Lactic acid bacteria (LAB) are commonly found in ecological niches rich in nutrients. The adaption to nutrient rich environments has resulted in extensive genome reduction and fastidious growth requirements including the demand for multiple amino acids . In nutrbtilisin .Lactococcus lactis cell envelope proteinases (CEP) have been extensively studied. The L. lactis CEP is a large multi domain enzyme covalently attached to the peptidoglycan layer at the outside of the cell wall . Domain S8 is found in 43564 sequences and a large class of those contain an insertion of a protease associated domain, PA(PF02225) within the S8 domain. Among those, there is often a fibronectin-like domain just downstream for the S8 domain. In prokaryotic proteases the fibronectin-like domain has been termed Fn3_5(PF06280). Fn3_5 is found in 1515 sequences and always together with S8. In plant proteases, the fibronectin-like domain is termed Fn3_6(PF17766) and this domain is found in 5862 sequences. The possible interaction between PA, S8, and Fn in the CEP of L. lactis might reveal a common principle used by this large class of proteases. The objective of this study is to use current state of the art structure prediction algorithms to establish a structural model of the CEP of L. lactis and to examine if the predicted model have implications for the function of the enzyme.The architecture of the prtP genes of Lactococcus lactis strains Wg2 and SK11 were the first CEP genes to be cloned and sequenced  are serine proteases with a catalytic triad consisting of three amino acids, aspartic acid, histidine, and serine. In the sequence of PrtP of MS22337 these amino acids are at the positions D215, H279, and S618. The relative positioning of these three amino acids in the six structures is given in By including the pro-peptide in the iTASSER modeling the two structures iTAS_3 and iTAS_4 were obtained as the two top ranking structures. The iTAS_3 model carries the pro-peptide as a long appendage whereas the pro-peptide in iTAS_4 is molded into the structure of the proteinase domain. The path of the pro-domain is passing the D and S of the catalytic triad in a distance of 4–8 Å from the amino acids of the catalytic triad. The path of the pro-peptide in this model seems to follow the catalytic cleft and thus defines the orientation of substrate bound to the protease. The pro-peptide is the very first substrate for the newly synthesized enzyme molecule and must therefore fit into the active site of the protease. In the iTAS_4 model the amino acid of the pro-peptide closest to the catalytic triad is residue number 161 whereas the activation is supposed to be a cleavage between residue 185 and 186. The possible interaction between substrate and the PrtP enzyme at domains further downstream has previously been suggested . From thMarinomonas primoryensis of which the structure has been determined and listed under the accession 4p99 in the PDB database. The reported structure is for a unit cell containing a tetramer of four antiparallel chains of identical structure. Each of the four chains contain four fibronectin like domains surrounded by calcium ions  prefer β-casein and all types cleave β- casein at multiple sites with no obvious specificity  growth rate in reconstituted milk of strains expressing the proteinase in question, (2) hydrolysis of intact αS1 casein and β- casein, (3) hydrolysis of chromogenic peptide substrates containing a p-nitroaniline at the C-terminal end, and (4) hydrolysis pattern of a 23 amino acid long peptide from the N-terminus of αS1 casein. A body of knowledge and a large number of enzymes with altered properties have been the result of this research. However, it seems that it has been somewhat elusive to get a firm grasp on the specificity of lactocepin. Maybe we need to make a slight shift in our approach to understand the specificity of this type of proteases. Understanding protease specificity based on the Schechter and Berger model for the active site of papain  (The CEP enzymes cleave only at very few positions within the peptide αS1(1–23) •The CEP enzymes are selective regarding which caseins to hydrolyze and they leave whey proteins unhydrolyzed .This seemingly contradictory behavior of an enzyme being unspecific and yet very selective can be reconciled if the enzyme is non-selective regarding the site cleaved but selective regarding the length or the nature of the substrate. An enzyme with these properties would also fit the needs of the bacterium for growth in milk.We find that the model emerging from our investigations seems to support such a mechanism:•The active site is similar to subtilisin which is a rather unspecific enzyme. However, in PrtP the enzyme is in the off position due to a slight displacement of the histidine of the catalytic triad•The catalytic cleft has at either site an obstruction which might function as push-buttons needing to be activated by substrate to activate and switch on the histidine. This feature might assure that short peptides are not being “over degraded.” Long peptides might be cleaved at several positions whereas short peptides are not cleaved at all.•Entire proteins might require auxiliary binding domains to allow for a bulky protein to squeeze into the catalytic cleft. This function might be served by the area on the surface of the fibronectin domains in the A-domain This model fits well also with the published protein engineering investigations.S1-casein or β- casein  at three sites between 16 and 23 with the site closest to the N-terminus being between 16 and 17. Mutant GLA is able to cleave closer to the N-terminus at bond 13–14, mutant GDT goes as close to the N-terminus as the Wg2 PrtP and cleaves at bond 8–9, whereas mutant GPP is changed in the other direction to give a longer product by only cleaving at one site between 21 and 22 , N166, S433, and K748 corresponding to position 322–324, 351, 618, and 933 in the MS22337 PrtP sequence. Except for the serine, which is the serine of the catalytic triad, all the positions could be mutated without losing the proteolytic activity of the enzyme. Change of the serine of the catalytic triad to an alanine led to complete inactivation of the enzyme. The AKT triplet could even be deleted or have insertions of 6 or 12 amino acids and still support growth with only minimal reduction in growth rate . The AKT1 and 22 .Lactococcus lactis is the need for a maturation protein PrtM for activity  can be found in the article.EH conceived the research strategy, conducted the modeling analyses, and drafted the manuscript. PM contributed with expertise on protein modeling particularly the use of evolutionary algorithms and participated in writing the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ribonuclease Dicer belongs to the family of RNase III endoribonucleases, the enzymes that specifically hydrolyze phosphodiester bonds found in double-stranded regions of RNAs. Dicer enzymes are mostly known for their essential role in the biogenesis of small regulatory RNAs. A typical Dicer-type RNase consists of a helicase domain, a domain of unknown function (DUF283), a PAZ (Piwi-Argonaute-Zwille) domain, two RNase III domains, and a double-stranded RNA binding domain; however, the domain composition of Dicers varies among species. Dicer and its homologues developed only in eukaryotes; nevertheless, the two enzymatic domains of Dicer, helicase and RNase III, display high sequence similarity to their prokaryotic orthologs. Evolutionary studies indicate that a combination of the helicase and RNase III domains in a single protein is a eukaryotic signature and is supposed to be one of the critical events that triggered the consolidation of the eukaryotic RNA interference. In this review, we provide the genetic insight into the domain organization and structure of Dicer proteins found in vertebrate and invertebrate animals, plants and fungi. We also discuss, in the context of the individual domains, domain deletion variants and partner proteins, a variety of Dicers’ functions not only related to small RNA biogenesis pathways. Xenopus ovary cDNA expression library for double-stranded RNA (dsRNA) binding proteins (dsRBPs) ..47]. ThiGiardia . Notablye miRNAs , while te miRNAs . Interescleavage .The PAZ domain has been initially suggested to mediate complex formation between the proteins from PPD and Dicer families . Later, Trypanosoma brucei, contain just two RNase III domains [S. cerevisiae does not have Dicer and an active RNAi pathway, other yeasts: Saccharomyces castellii and Candida albicans [Kluyveromyces polysporus [E. coli RNAse III comprising one RNase III domain and one dsRBD domain [The simplest Dicer-type proteins, such as Dicer-like1 protein found in the early diverging protozoan  domains . Althougalbicans , and Klulysporus , do havelysporus . A commolysporus . In the lysporus ,49,102. lysporus . It was lysporus . Moreovelysporus . These rlysporus . In the lysporus . Intereslysporus . The ~10D domain .The pattern of recognition and cleavage by plant DCL proteins differ from the mechanisms observed for animal Dicers. The plant miRNA precursors are much more variable in size and shape than the animal miRNA precursors and, importantly, they are completely processed in the nucleus by DCL1 ,107,108.2+) important for catalytic activity, or stabilization the dimeric structure of the enzyme [RNase III enzymes are highly conserved in the Bacteria and Eukarya, however, are only sporadically observed in the Archaea . Simple e enzyme ,116,117.e enzyme . Importae enzyme ,119.E. coli RNase III [The mechanisms of substrate recognition and cleavage by RNase III orthologs have been provided by studies on Nase III ,120. PreNase III .The dsRBD domain is a small conserved protein domain found in eukaryotic, prokaryotic and viral proteins F; probabE. coli RNase III, form a homodimer to generate ~11-bp products from dsRNAs [The central property of dsRBD is to bind to dsRNA, and proteins that include this domain were found to be involved in many aspects of cellular life, including RNA editing, RNA processing, RNA metabolism, RNA transport, RNA silencing, and even antiviral response . Originam dsRNAs . The prom dsRNAs . Interesm dsRNAs . The addm dsRNAs .Schizosaccharomyces pombe Dicer is also critical for the nuclear localization of the protein. In contrast to hDicer, S. pombe Dicer localizes predominantly in the nucleus, near the nuclear pore complexes [S. pombe Dicer has a C-terminal extension composed of an α-helical turn followed by a zinc-coordination motif [S. pombe Dicer. Interestingly, the extended dsRBD of the S. pombe Dicer is a temperature sensitive domain which acts as a thermo-switch. When the temperature rises above 34 °C, the nuclear retention properties of the dsRBD changes, and the S. pombe Dicer moves to the cytoplasm, with important consequence for stress-dependent gene regulation and response [Mammalian Dicers predominantly localize to the cytoplasm to perform pre-miRNA cleavage, but several reports have provided evidence for nuclear function and localization of these proteins ,127. Theomplexes . It was on motif . This stresponse .A. thaliana suggest that DCL proteins could be involved in tRF biogenesis in plants [Drosophila and S. pombe revealed that the lack of Dicer does not significantly affect tRF levels [In addition to miRNA and siRNA production, Dicer has been implicated in the biogenesis of a group of small RNAs, which are derived from tRNAs, called tRNA-derived fragments (tRFs) . Studiesn plants . HoweverF levels . Some tRF levels .A. thaliana in DCL2, DCL3 and DCL4 mutants [Beyond the biogenesis of small regulatory RNAs, Dicer also performs functions that are related to its nuclear localization. In organisms such as flies, plants and fission yeast, nuclear Dicer is involved in formation of heterochromatin . In addi mutants . Interes mutants . Nuclear mutants . This wa mutants .C. elegans the apoptosis-induced caspases can cleave Dicer within the RNase IIIa domain, which results in releasing the C-terminal fragment of Dicer. Such truncated Dicer does not act as an RNase III enzyme; instead it displays the activity characteristic of DNases by initiating the chromosome fragmentation in the nucleus [Dicer ribonuclease was found to be directly involved in the apoptosis process . Apoptos nucleus . Such DN nucleus .Domain fusions are a common occurrence throughout protein evolution . In the G. intestinalis has only PAZ and two RNase III domains, which are entirely sufficient to specifically recognize and bind dsRNA and generate small RNAs of a certain length (~25-nt). Gaining the helicase domain has allowed Dicer-type proteins to serve a function of a cytosolic sensor of viral RNAs. However, the helicase domain of vertebrate Dicers seems not to be involved in the viral RNA recognition process. Instead, it provides another layer of regulation of substrate recognition by discriminating among pre-miRNA substrates, and between pre-miRNAs and pre-siRNAs. Consequently, gaining the helicase domain by ancestor Dicers has allowed to consolidate the cleavage, sensor and regulator activities in one protein [C. elegans Dicer by death-promoting caspases triggers the rearrangement of the remaining C-terminal fragment, containing one active RNsase III domain and one dsRBD domain. Such truncated C. elegans Dicer lacks the RNase III activity, but instead it benefits from being a DNase [Gain and loss of protein domains are important events that have been occurring abundantly during evolution of species. A minimal Dicer from  protein . Moreove protein  has allo protein ,72,73, a protein ,131. Mor protein ,129. Reg length ~-nt. Gain a DNase . There i


This study deals a phenomenon of magnetic domain transition for the stepped magneto-impedance element. Our previous research shows that an element with 70° inclined easy axis has a typical characteristic of the domain transition, and the transition can be controlled by the normal magnetic field. In this paper, we apply this phenomenon and controlling method to the line arrangement adjacent to many body elements, in which mutual magnetic interaction exists. The result shows that the hidden inclined Landau–Lifshitz domain appears by applying a distributed normal field the same as an individual element. Hc [The observation of a magnetic domain of a thin film magnetic element is carried out using measuring methods such as electron beam , Kerr maHc , direct Hc , and a bHc . A domaiHc ,21.The stepped-MI element has the unique property of a step-like magneto-impedance change, in the case where the sensor has an in-plane uniaxial inclined easy axis ,23. A doIn this study, an extension of the application of this phenomenon to clustered many-body elements was experimentally investigated. The individual element has the same width and thickness, and also has the direction of magnetic anisotropy the same as an element having the stepped-MI property with a hidden ILLD state. A certain number of the elements were arranged in a planar cluster which has a configuration of a line arrangement adjacent to many-body elements. This trial is important for realizing a high-density device using this phenomenon. The magnetic hysteresis loop (MH-loop) of the planar clustered element was measured in accordance with a domain observation. An effect of the application of the distributed normal field was also investigated.85Nb12Zr3 film was RF-sputter deposited onto a soda glass substrate and then micro-fabricated into rectangular elements by a lift-off process. The element was thousands of µm in length, 20 µm wide, and 2.1 µm thick. A uniaxial magnetic anisotropy was induced by magnetic field annealing, 240 kA/m at 673 K for 1 h. The easy axis of the magnetic anisotropy is made along the processing magnetic field. The annealing apparatus used in this study had an accuracy in the angle position of 0.5°. In this study it was induced in several different conditions for a comparison of their properties.The element was fabricated by a thin-film process. An amorphous Coθ = 70°. The element dimensions were as follows: In the case of the adjacent many-body elements, the length was 3000 µm, the width was 20 µm, and the thickness was 2.1 µm. The element length was determined by a knowledge obtained by the previous study that the element would have a residual domain at the both end of element. In our previous report, the element length was set as 2000 µm [θ = 61°, θ = 71°, and θ = 90°. Our previous study showed that the element of θ = 61° has a changing property from single domain, −, to the ILLD and then to single domain, +, with the increasing magnetic field. The element of θ = 90° has a changing property between the single domain − and +. These are the neighboring conditions in the direction of easy axis in θ = 71°, which is the focusing condition in this study. The element layout on the glass substrate is shown in θ = 67° against the width direction of the element strips. This layout on the glass substrate is shown in θ = 67° was expected to have a property of the three stable states [We made two different layouts of the elements. One was the configuration of the line arrangement adjacent many-body elements having a mutual magnetic interaction with each other, and the other layout were dispersed individual elements. These two types of different layouts were made for a comparison between the elements having the mutual force and those without it. The prior many-body element was prepared in three different conditions for the purpose of confirming the effect of the direction of easy axis with a focus on the element having the hidden domain state, which is around  2000 µm . This ele states  with a nThe measurements carried out here were a MH-loop measurement and a magnetic domain observation. The MH-loop was measured by a vibrating sample magnetometer (VSM) . The magnetic domain was observed by a Kerr microscope  by applying a magnetic field in a certain direction and also at a certain controlled strength. These applied magnetic fields were generated by the following two apparatuses. The distributed normal field was made using a ring-shaped magnet which was positioned below the observation stage of the Kerr microscope. The magnetic field along the element’s longitudinal in-plane direction was controlled using a Helmholtz coil  while the observation of the magnetic domain was carried out. The definition of the distributed normal field is shown in A photo of the fabricated many-body elements on a glass substrate are shown in θ = 61°, θ = 71°, and θ = 90°. The measurement sweep speed was 0.1 Oe/s (7.96 (A/m)/s) and the measurement time constant was 100 ms. The MH-loop in each condition resembles each other. The coercivity slightly increased with the increase in the θ value, and the linearly-inclined part of the curve changes to a slightly bending configuration in the vicinity of the zero field with the increase in the θ value. In these figures, the value of Meff (T) is estimated from the volume 3000 μm × 3020 μm × 2.1 μm. This is an effective value, because it is estimated as if the element has a shape of a single square sheet which corresponds to the volume of many-body elements as a whole.Firstly, the MH-loop measurement for the many-body elements with different directions of the easy axis was carried out. The length direction of the element line was set in the measurement direction of the VSM apparatus. Measured MH-loops are shown in θ = 61°, θ = 71°, and θ = 90°. The elements were magnetically saturated in the value around 300–400 A/m. Based on our previous study the variation of the magnetic domain for the individual single element, having an easy axis at θ = 61°, has a property of domain variation which changes from a single domain in the − direction to ILLD around zero magnetic field, and then a single domain in the + direction, with the increase in the magnetic field from minus value to the plus value. In the case of θ = 71°, it is the element having the hidden-stable state. This means that the element has an energetically stable ILLD state, however, it does not appear using a method of application of a uniform external field. Apparently the domain switched between the − single domain and the + single domain for the θ = 71° element. In the case of θ = 90°, the domain simply switched between the − single domain and the + single domain. The measured result of this study, as shown in the (a)θ = 61°: The initial single domain in the − direction of whole elements as shown in the photo view change to the ILLD gradually and individually with the increase in the applied field. After the domain of all elements changes to the ILLD, then the width of the area of the striped domain gradually changes and, finally, the stripe of the ILLD is erased and all elements change to the single domain in the + direction. When the elements are all in the state of a single domain, +, the many-body element is getting magnetically saturated.(b)θ = 71°: The initial single domain in the − direction of whole elements as shown in the photo view change to the single domain in + gradually and individually with the increase in the applied field. When the elements are all in the state of single domain +, the many-body element is getting magnetically saturated.(c)θ = 90°: The domain transition was the same as the one with θ = 71°.The observation of the magnetic domain was also carried out and shown in From the view point of the magnetic domain, both θ = 71°, and θ = 90°. The measured MH-loop in For the purpose of discovering the effect of magnetic mutual interaction, a comparison of the magnetization process between the adjacent many-body element and the dispersed individual elements was carried out based on the variation of the domain transition number as a function of the applied field, as plotted in θ = 67° element has a splitting appearance of the ILLD as a function of the applied field. When the field is increasing the − single domain switched to the ILLD in the + region of the applied field, and it switches within the − region when the field is decreasing. This profile was already reported and explained previously [eviously . In FiguBased on θ = 71° was experimentally shown to behave as if it is the individual element having the same easy axis direction. The domain of the consisted each element switched as expected from the behavior of the individual single element with hidden ILLD state, whereas the domain switching does not occur simultaneously within the many-body element. This dispersed behavior in the switched field would be expected to come from the mutual magnetic interaction. In this section, an application of the distributed normal field was tried for the θ = 71° many-body element.From here an examination of the reconstruction of the hidden ILLD is shown. The method used here was previously developed by us  for the xB as a function of the X-position at different distances from the magnet, d = 8.5 mm and d = 10 mm. “d” was defined in x = 0 was placed on the center axis of the ring-shaped magnet, and the measured element was placed on the X–Y plane at a certain Z-position, which means different d-positions in this measurement. The variations in the figure show linear configurations having different inclination, one is xB (mT) = 0.54 × x (mm) at d = 10 mm and the other is xB (mT) = 1.22 × x (mm) at d = 8.5 mm. The normal field zB as a function of X-position is shown in x = 0 was 11.5 mT at d = 10 mm and 8.7 mT at d = 8.5 mm. The normal field in the element’s rectangular area varies at most 8%, therefore, it has a slight difference from the ideal variation , and d = 8.5 mm in which the distributed parameter x/∆x∆B = 1.22 (T/m). These results show that the ILLD state appears in the central area of the many-body element when the distributed normal field is applied. These ILLD have a certain periodical pattern in the element lines in spite of the randomly-switched single domain appearance at the zero field without the distributed normal field. This periodical pattern has a variation as a function of the distributed parameter.The result of the application of the distributed normal field to the element having the easy axis in A consideration concerning the magnetic mutual interaction within the many-body element is discussed here. A qualitative estimation of the interaction is carried out based on a magnetic field distribution originating from one narrow magnetic strip which is a construction element of the many-body element.sM = 0.93 T. This sM value was the measured value of Co85Nb12Zr3 amorphous thin film. eD.xB, which is the field in the direction of longitudinal axis is shown as a parameter of element distance eD. The horizontal axis of the figure shows a longitudinal position x on the axis of the neighboring virtual element. The direction of magnetic field xB has a negative value, due to the magnetized direction of the existing element being positive, as shown in xB. The element distance eD ranges from 40 µm to 200 µm, which corresponds to the position from the just neighboring to the 5th neighboring element. xB| has a maximum value in the vicinity of the end of the element, the range was almost 0.3 mm from the end, and the maximum value rapidly decreased with the increase in the eD value. From −4 T). The magnetic field which exceeds this value would expect to have a promoting effect of domain transition. The inverse field generated by a transited element have a blocking effect for the neighboring elements. The effect is expected to be strongly affected to the 1st, 2nd, and 3rd neighboring elements.xB on the Y-axis of eD. The xB monotonically decreases as a function of eD, such as xB = 2.7 × 10−6 T (27 mG) at eD = 20 µm and xB = 1.0 × 10−6 T (10 mG) at eD = 1.5 mm. This magnetic field is much smaller than the maximum value of the one in −4 T).The magnetic field distribution in According to the experimental observation in this study, the blocking effect of the domain transition in the neighboring element of a transited single narrow element was observed, as can be seen in θ = 71°. The MH-loop of the planar clustered element was measured in accordance with a domain observation. The MH-loop of the clustered many-body element which has the easy axis in θ = 71° was almost the same as the one having the easy axis in θ = 61° and θ = 90°. The each element has suffered a mutual magnetic interaction between the consisting element strips, as if it suffered the demagnetizing force. The variation of magnetic domain for the θ = 71° many-body element was a gradually changed from the number of switched elements having the single domain, and which is the same as the domain variation of θ = 90°. An effect of the application of the distributed normal field was also investigated. The ILLD appeared by applying the distributed normal field. A periodicity of the domain distribution formed by the consisting elements was observed, and the periodical pattern changed as a function of the distributed parameter ∆Bx/∆x.An investigation of the stepped-MI phenomenon for the clustered many-body elements was experimentally carried out. The 76 elements were arranged in a planar cluster with the line/space = 20 µm/20 µm having a configuration of the line arrangement adjacent to many-body elements. The individual element in it has the same width and thickness, and also has the direction of magnetic anisotropy the same as an element having the stepped-MI property with a hidden ILLD state, which is


Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined.Zinc is an essential nutrient for the virulence of bacterial pathogens such as  Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes.Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of  In prokaryotes, Zn2+ serves as a crucial structural and/or catalytic cofactor in ca. 5 to 6% of the proteome and is involved in critical processes such as carbon metabolism, competence, and the regulation of DNA transcription  family of importers  is the leading cause of bacterial pneumonia, which accounts for 15% of all childhood disease mortalities, and has a global economic health burden of more than US$4 billion annually  that is structurally related to ZinT, a periplasmic protein from E. coli and Salmonella  by an 11-amino-acid linking region . His-rich loops are prevalent in Zn2+-specific SBPs, although their contribution to Zn2+ recruitment also remains poorly defined  simulations, and electron paramagnetic resonance (EPR) studies to elucidate the molecular basis of Zn2+ acquisition in AdcA. We report the first high-resolution structure of a Zn2+-bound two-domain SBP, AdcA, and reveal the essential role of the AdcAN domain in pneumococcal Zn2+ uptake and the relative contributions of the “accessory” regions (His-rich loop and AdcAC domain) to this process. We then investigate the conformational landscape of the AdcAN domain and show that ligand-binding induces only localized structural rearrangements within this domain. A key structural element within the AdcAN domain is surface loop α2β2, a highly dynamic region that is stabilized by ligand-binding in a process crucial for AdcCB-mediated Zn2+ uptake. Collectively, these findings provide new mechanistic insight into how Zn2+-specific cluster A-I SBPs bind metal ligands and the structural elements that contribute to bacterial Zn2+ import. These findings provide a structural framework for the development of novel inhibitors targeting AdcA against pneumococcal infections.To date, high-resolution crystal structures have only been determined for single domain SBPs from Gram-negative organisms, while two-domain SBPs have remained refractory to such approaches , 23, 24.2+-bound AdcA was determined at 1.58-Å resolution and revealed a two-domain organization with the AdcAN and AdcAC domains forming discrete globular regions, connected by an 11-amino-acid linker and each containing one Zn2+ ion 4 lobes bisected by a cleft in which the metal binds. The binding site in AdcAN is comprised of three Nε2 atoms from His63, His140, and His204 and one Oε2 atom from Glu279. These four residues bind a single Zn2+ ion via 4-coordinate geometry, with bond distances of 1.98 to 2.08 Å  showing that other divalent first-row transition metal ions did not induce stabilization of the N-terminal domain . This fold is highly conserved among cluster A-I SBPs, including S. pneumoniae AdcAII , Salmonella enterica ZnuA , and S. pneumoniae PsaA   remaining the same during the five independent MD simulations. This partial open conformation was also observed in the S. enterica Zn2+-bound ZnuA structures . The high-resolution crystal structure of Zn2+-bound truncated AdcAN (Electron density was not observed for 14 residues (120 to 133) of the His-rich loop, consistent with the predicted flexibility of this region , 27. Theormation , 22, 26.ed AdcAN  did not C domain has a lipocalin-like fold of an 8-stranded up-and-down β-barrel (β9-16) and a helical region of four short helices (α11 to α15)  and S. enterica  . The high-resolution crystal structure of isolated Zn2+-bound truncated AdcAC did not reveal any conformational differences by comparison with the domain present in the full-length protein  . The Zn2o 2.11 Å . These bhan Zn2+ . The ove8 Å) see . The str protein . The strn  Structural differences between MD simulations. (B) Modeling DEER distance distributions. (C) Instrument parameters and data analysis. Download Copyright © 2021 Luo et al.2021Luo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01958-20.3TABLE S2N, and AdcAC. (B) Residue pairs forming the interface between AdcAN and AdcAC. (C) Hydrogen bond analysis of the lobe-linking α-helix. Download Table S2, PDF file, 0.1 MB.(A) Cα RMSD values of AdcA, AdcACopyright © 2021 Luo et al.2021Luo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+-bound AdcA, interaction between the domains occurs between α12 and α15 of the AdcAC domain and α7 and the base portion of the His-rich loop (residues 109 to 117) of the AdcAN domain, with a buried surface area of ∼1,200 Å2. An extensive array of intramolecular interactions was observed at the interface of the two domains. MD analyses suggest that the most stable portion of the interface arises from interactions between six residues from each domain. The most stable part of the interface involves the salt bridge between Lys368 and Asp213 and H-bonds formed by the side chain of Tyr365 with the backbone and side chain of Asp213 and the backbone of Leu212. Residues Gln492 and Gln496 also form a stable part of the interface, but their interacting residues are dependent upon protein conformation. Gln492 alternates between forming H-bonds with the backbone of Leu115 and the side chain of Asp112. The side chain of Gln496 interacts with both the aromatic ring of Phe137 and the backbone of Leu115. Similarly, Glu120 in the AdcAN domain forms a salt bridge with Lys318, as well as H-bonds with the imidazole ring of His501 in the AdcAC domain, depending on the conformation of the protein. The interactions between these residues define a relatively large and stable buried surface area via a combination of salt bridges, H bonding, and transient interactions. The stability of these interactions and average distances are provided in 2+ acquisition.In ZnN domain was investigated using mutant variants of AdcA wherein the N-terminal domain was deleted (adcAC) or the Zn2+-coordinating His residues  were mutated to Ala (adcAΔHis), thereby abolishing its Zn2+-binding capacity. The contribution of AdcAN to Zn2+ uptake was then assessed in the S. pneumoniae ΔadcAII background. The resultant strains (ΔadcAII ΔadcA::adcAC and ΔadcAII ΔadcA::adcAΔHis) showed a significant growth defect in Zn2+-restricted conditions  and had a growth profile in Zn2+-replete media similar to that of the ΔadcA ΔadcAII deletion strain, wherein Adc permease function is abrogated  in Zn2+ uptake. AdcA variants were generated in which (i) the AdcAC domain was truncated (adcAN), (ii) the His-rich loop was deleted (adcAΔLoop), or (iii) both AdcAC and the His-rich loop were deleted (adcANΔLoop). In the ΔadcAII background, deletion of either accessory region had no significant impact on growth in Zn2+-restricted conditions, compared to Zn2+-replete conditions (adcANΔLoop strain) resulted in a significant growth defect under Zn2+-restriction  ion-recruiting SBP PsaA, for which a ligand-binding mechanism and the relationship of this mechanism to cation import have been reported , revealed that they did not exhibit high RMSF values. Further, examination of the H-bond network of the lobe-linking α-helix  region in Zn2+-bound AdcAN showed that all H-bond lengths were ∼3 Å, the ideal range for a helical conformation  . Formatireported , 28. In reported . Given tormation , and theormation . Thus, tN domain was then directly examined using double electron-electron resonance (DEER) spectroscopy. Five full-length AdcA variants were generated, by combining pairs of Cys residues in the N-terminal domain of the protein, each of which was analyzed in the metal-free and Zn2+-bound states  in the mean distance distributions, as well as small changes in the distance distribution widths  as a function of time in the presence and absence of Zn2+ (2+-bound states of AdcA (2+-bound states (2+-bound states. Loops α10β8 (Glu279) and α4β4 (His140) showed low mobility in either state. These data suggest that His140, His204, and Glu279 provide a largely preformed metal-binding site, with loop α2β2 (His63) acting as a region of dynamic mobility that is stabilized by Zn2+ binding.The loops containing the metal-coordinating residues are the regions in the AdcA of Zn2+ . This re of AdcA . Furtherd states . In contT60C (located on loop α2β2) and AdcAT98C (located on helix α3), were examined in the presence and absence of Zn2+. We observed that the MTSSL label was more mobile in AdcAT60C, by comparison with AdcAT98C  in facilitating bacterial metal uptake. In AdcA, the solvent-accessible accessory regions, i.e. the His-rich loop and the AdcAC domain, serve to aid in Zn2+ recruitment from bulk solvent and increase its availability to AdcAN. Ligand binding in the AdcAN domain occurs at a preformed, relatively static metal-binding site and, although not associated with global changes in protein conformation, stabilizes the surface loop α2β2. In metal-free AdcA, loop α2β2 is highly mobile and increases the solvent accessibility of the metal-binding site. Upon interaction with Zn2+, the metal-coordinating residue His63, contained within loop α2β2, initiates closure of the metal-binding site as the loop is pulled onto the binding site, thereby decreasing solvent accessibility. The concomitant reduction in loop α2β2 mobility stabilizes the AdcAN surface. Zinc-bound AdcA is then able to productively interact with the cognate AdcCB transporter and release the bound metal into the translocation pathway. The molecular details of the interaction between AdcA and AdcB and how Zn2+ is released to the transporter remain to be elucidated. These structural features, which can be summarized as a limited conformational landscape, a rigid metal-binding site, and a flexible surface loop, appear to be well conserved among Zn2+-specific SBPs, suggesting a common mechanistic basis for prokaryotic Zn2+ uptake  Oligonucleotide primers used in this study. (B) Strains used in this study. (C) Plasmids used in this study. Download Copyright © 2021 Luo et al.2021Luo et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli LEMO21(DE3) and purified essentially as described previously  experiments were performed in technical triplicate in at least four independent experiments to determine the mean melting transition (mT) (mT ± (SEM) were calculated using GraphPad Prism (v7.0d). Affinity determination of the mutant AdcAN variants was performed essentially as described previously  . Fluoreseviously .2+-bound AdcA were obtained in 10% (wt/vol) polyethylene glycol (PEG) 20000, 18% (vol/vol) PEG monomethyl ether (MME) 550, 0.03 M CaCl2, 0.03 M MgCl2, and 0.1 M MES/imidazole (pH 6.5) at 291 K, with a protein concentration of 10 mg ml−1 and ZnCl2 at a 1:10 protein/Zn2+ molar ratio, using the hanging-drop vapor diffusion method. The AdcAN domain fragment was crystallized as described previously  and 30% (wt/vol) PEG MME 5000 at 293 K. Additional details are provided in Protein crystals of Zneviously , and theS-methyl methanesolfonothionate (MTSSL). Free MTSSL was removed by dialysis (10-kDa MWCO) in 1 liter of buffer solution  at 277 K for 24 h. The dialyzed sample was concentrated to 500 μl (10-kDa MWCO) and purified (Superdex 75 Increase 10/300 column). The sample was concentrated to 100 μM (10-kDa MWCO) and flash-frozen (liquid N2). X-band CW (continuous wave) EPR spectra in solution were measured on a Bruker Elex E540 spectrometer equipped with a Bruker Super High Sensitivity resonator and a liquid N2 temperature control system. Instrument parameters are described in In silico modeling of the spin label rotamer distributions for the metal-free and Zn2+-bound protein conformations was computed using MMM 2018.2 (Labeling of the AdcA-Cys variants (10 μM) was performed by incubation with 100 μM M 2018.2 .2+-bound AdcA was used as the starting structure for all simulations with the set up and parameterization details described in The crystal structure of ZnA73C/A259C used the maleimide derivatives of dyes Alexa-555 and Alexa-647, with further details provided in A73C/A259C (25 to 100 pM) was studied with smFRET/ALEX at room temperature . All experiments were performed using a bespoke confocal microscope assembly as detailed by Husada et al. , 7JJ8 (Zn2+-bound AdcAN), 7JJA , and 7JJB (Zn2+-bound AdcAC).The accession codes for the structures deposited in the Protein Data Bank are as follows:


The relative tail pressure with subadditive potentials is introduced via open random covers for continuous bundle random transformations. A variational principle is established and the defined pressure turns out to be invariant under the principal extension. The topological pressure with additive potential was introduced by Ruelle and Walters ,2. The tThe topological pressure with zero potential reduces to the classical topological entropy. For the purpose of measuring the local complexity of compact dynamical systems at arbitrary small scales, Misiurewicz introduced topological tail entropy  for contMa et al. set up a relative tail maximal entropy principle  and a taIn this paper we introduce the relative tail pressure with subadditive potentials for continuous bundle random transformations via open random sets. The notion is a little different from that developed before ,30 for tOrganization of the paper is as follows?We recall some basics of the relativized ergodic theory in In this section, we recall some basic notions of the relative measure-theoretic entropies for bundle random transformations ,26. For X be a compact metric space and X. A continuous bundle random dynamical system (RDS) T over Let Let Let The relative entropy of Let eference  for the For any The existence of the limit follows from the well-known subadditive argument. Q is measurable, where X. The map U in The map For each For each g on By replacing the function eference  with fn,Lemma 1.Ω to The map The limiteference ,35). LetNotice that In this section we consider the relationship between the relative tail pressure, Lyapunov exponent with subadditive potentials and the relative entropy over the measurable subset of the product space We first give the power rules for the relative conditional pressure and relative tail pressure with subadditive potentials in the original continuous bundle RDS.Proposition 1.Θ be a skew product transformation, Φ be subadditive and Let Proof. Let Clearly,ThenSince Thus Proposition 2.Θ be a skew product transformation and Φ be subadditive. Then Let Proof. By Proposition 1,Since S over Consider another compact space Definition 1.A continuous bundle RDS T is called a factor of another continuous bundle RDS S if a family of continuous surjective maps We now take up the consideration of the measurable subset Let For the given the We need the following two important Lemmas. The first Lemma shows the upper semi-continuity of the conditional entropy, which can be found in many references, for instance ,7. The sLemma 2.Let Lemma 3.Let where For any given finite measurable partition of the original RDS, we give an inequality relating the relative conditional pressure with subadditive potentials and the relative entropy of the product RDS with respect to invariant measures.Proposition 3.Γ be the skew product transformation on Φ be subadditive with Let Proof. Let y V hμ and andΓm, ΘThe following theorem describes the variational inequality between the relative tail pressure with subadditive potentials, the defect of upper semi-continuity of the relative entropy function and Lyapunov exponent with subadditive potentials with respect to invariant measures.Theorem 1.Γ be the skew product transformation on Φ with Let Proof. Let ThenBy the arbitrariness of the partition In this section we investigate the variational principle between the defect of upper semi-continuity of the relative entropy function of the self-product RDSs and the relative tail pressure with subadditive potentials of the original RDS.Denote by We will use the following Lemma  in the pLemma 4.Let Moreover, For any given open random cover of the original RDS, the following construction of a maximal invariant measure sets up a relationship between the relative conditional pressure with subadditive potentials and the relative entropy of the self-product RDSs, which is essential for the argument of the variational principle.Proposition 4.Θ be the skew product transformation on Φ be subadditive and Let (i) (ii) the support of Proof. Choose some Fix Notice that Let It follows from Theorem 1.5.8 in Reference  that theWe now show that For the first proposition, let Let Since different elements of It is not hard to verify thatNotice thatThenj with For each j, Since the entropy function It follows the inequality  that1mHμConsidering the selected subsequence By taking Choose a refine sequence of eference  we havehFor the second proposition, we omit the argument since it is very similar to that of Proposition 4.2 in Reference  and we cWe now show that the relative tail pressure with subadditive potentials of the original RDS could be reached by the defect of the upper semi-continuity of the relative entropy together with the Lyapunov exponent of the subadditive potentials with respect to some invariant measure of the self-product RDS.Proposition 5.Θ be a skew product transformation on Φ be subadditive with Let Proof. Let m with By ( Lemma 2.Notice thati, Let Since It follows directly from Theorem 1 and Proposition 5 that the desired variational principle holds.Theorem 2.Θ be a skew product transformation on Φ be subadditive with Let Let Definition 2.Λ is called a principal extension of the skew product transformation Θ if the relative entropy A skew product transformation The following theorem shows that the relative tail pressure with subadditive potentials is invariant under principal extensions. The proof is similar to Theorem 4.3 in Reference  and we oTheorem 3.Φ be subadditive with Λ is a principal extension of Θ, then Λ and Θ and Let


Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors. RNA-dependent RNA polymerases from segmented negative stranded RNA viruses catalyze genome replication and viral transcription. Here, the authors present the cryo-EM structure of full-length La Crosse virus polymerase and structurally characterize the pre-initiation and elongation states, which is of interest for the development of polymerase inhibitors. Bunyavirales is a very large and diverse order of segmented negative-strand RNA viruses (sNSV) comprising more than 500 species classified into 12 families1. It contains serious human pathogens such as La Crosse virus , Hantaan virus , Crimean-Congo hemorrhagic fever virus , Rift Valley Fever virus , and Lassa fever virus . Viruses from the Bunyavirales order are related to other sNSV and in particular to influenza virus, a major human pathogen belonging to the Orthomyxoviridae family.Bunyavirales2. These processes are performed in the cytoplasm of infected cells for Bunyaviruses, whereas they occur in the nucleus for influenza virus4. Replication generates full-length genome or antigenome copies , whereas transcription produces capped viral mRNA that are recognized by the cellular translation machinery to produce viral proteins. Transcription is initiated by a “cap-snatching” mechanism, whereby host 5′ capped RNAs are bound by the L cap-binding domain (CBD), cleaved by the L endonuclease domain several nucleotides downstream, and then used to prime synthesis of mRNA6.Replication and transcription of sNSV viral genomic segments are performed by the virally encoded RNA-dependent RNA polymerase, also called L protein for 7, bunyavirus polymerases act in the cytoplasm. It is currently unclear which cytoplasmic capped RNAs are accessed by bunyavirus polymerases, and in what context, and if the polymerase contains specific domains that interact with host capped-RNA-bound proteins. Second, the length of the host-derived capped RNA primer generated after cleavage by the endonuclease differs between families5, 0–7 in Arenaviridae, 10–18 in Peribunyaviridae, 10–14 in Orthomyxoviridae, suggesting differences in the relative position of the endonuclease, CBD and polymerase active site. Third, CBD localization within L proteins remains unclear for several viral families primarily due to the absence of a definitive motif for the cap-binding site and because of the high divergence in sequence between polymerases, particularly in their C-terminal region. Identification of the CBD in the C-terminal region of L proteins has however recently been achieved for both Rift Valley Fever virus  and California Academy of Science virus , thanks to the determination of isolated CBD domain structures9.Although the overall mechanism of transcription initiation is likely conserved between sNSVs, several elements suggest some divergence between viral families. First, the source of capped RNA differs. Whereas influenza polymerase interacts directly with the host RNA polymerase II to snatch the caps of nascent transcripts in the nucleus12. A snapshot of transcription elongation has been captured revealing the presence of a nine-base-pair template-product RNA inside the active site cavity that is then separated into two single-stranded RNAs exiting through separated tunnels12. In comparison, structural information on Bunyavirales polymerase remains limited with only the structure of a C-terminally truncated construct of LACV polymerase  being currently available13. LACV-L1-1750 is composed of an N-terminal protruding endonuclease domain (residues 1–185) and a polymerase core containing the RNA synthesis active site (residues 186–1750). It was solved by X-ray crystallography in the pre-initiation state in complex with the 5′ and 3′ promoter ends. Both promoter ends bind sequence-specifically in separate pockets away from the active site, respectively, called the “5′ end stem-loop pocket” and the “3′ end pre-initiation pocket”. The LACV-L1-1750 structure also depicts the presence of an active site cavity with typical polymerase motifs as well as distinct template and product exit tunnels.To understand the detailed mechanisms of replication and transcription, structures of the full-length polymerase are essential. Significant advances have recently been made on influenza polymerase with structures stalled at different steps of transcription now being available. They reveal that influenza CBD undergoes a 70° movement to bring the capped RNA from an orientation in which it can be cleaved by the endonuclease to one where it can enter the polymerase active siteTo reveal the structure of the C-terminal region of LACV-L and the overall architecture of the complete polymerase, we determined the structure of full length LACV-L (LACV-L FL) by X-ray crystallography and high-resolution cryo-EM. We uncover the structure of LACV-L CBD, which contains a specific insertion allowing interaction with the endonuclease. We find that the extreme C-terminal region of LACV-L FL is a zinc-binding domain (ZBD) that is absent in other sNSV polymerases of known structure and may correspond to a host–protein interaction platform. We also capture snapshots of LACV polymerase in both pre-initiation and elongation-mimicking states, thereby revealing, amongst other conformational changes, the movement of the priming loop that unblocks the active site cavity and permits accommodation of a 10-base-pair template-product duplex, characteristic of elongation.13  but the endonuclease domain undergoes a large rotational movement of 180°  is conserved compared to the LACV-LThe endonuclease is held in place by hydrophobic interactions with a large number of residues from different domains Fig. . The N-tPeribunyaviridae L proteins, although there is no density for them. The m7GTP-binding sites of RVFV and influenza virus CBD14 which share the same overall fold are located in an equivalent loop  packed against the α-helix 77 that is flanked by a three-stranded antiparallel β-sheet , the α-helix 78 and long loops Fig. . There iThe CBD is rotationally mobile as visualized in a 3D variability analysis of the dataset  the nucleotide to be incorporated, (ii) the product RNA to be elongated, (iii) two magnesium ions, and (iv) the three aspartic acids of motifs A and C is necessary for catalysis of the phosphoryl transfer reaction, the organization of the active site changes subsequently20. Such post-incorporation structural changes are visible in LACV-L elongation mimicking stage. Perhaps related to the fact that there is no pyrophosphate (since there was no reaction), the two magnesium ions that control nucleotide addition are not present in the catalytic configuration. Instead, a presumed Mg2+ ion, coordinated by residues D1188 (motif C), E1237 (motif E), and the carbonyl oxygen of A1059 (motif A) is present, typical of the inactive open state of the polymerase active site  is an essential element that usually stabilizes the first ‘priming’ nucleotide of the product during replication initiation. In the pre-initiation structure, this loop protrudes towards the active site but is disordered probably due to the absence of RNA and nucleotides Fig. . As partThe transition from pre-initiation to elongation is also coupled to coordinated domain movements. The lid domain rotates by 12° compared to the thumb ring, resulting in the opening of the template and the product exit tunnels Fig. . SeparatPhenuiviridae, Arenaviridae, and Orthomyxoviridae polymerases. LACV-L CBD shares a conserved fold with equivalent structures from RVFV (Phenuiviridae)8, CASV (Arenaviridae)9, and influenza virus (Orthomyxoviridae)14, consisting of an antiparallel β-sheet stacked against an α-helix . In addition, LACV-L CBD contains a family-specific insertion consisting of a three-stranded β-sheet , α-helix 78 and charged loops (1932–1936 and 1956–1963). This insertion is likely related to the role of the CBD in transcription initiation as it is involved in interactions with the endonuclease , whereas the distal C-terminal region differs between the three families. Orthomyxoviridae and Arenaviridae, respectively, have a PB2 627-domain and D1-III domain that are structurally related11, whereas the ZBD present in LACV has a different fold. This suggests that Orthomyxoviridae and Arenaviridae polymerases are more closely related . The presence of the ZBD or PB2 627-domain in the C-terminal region in a protruding position compared to the polymerase core suggests that, as for the PB2 627-domain21, LACV-L ZBD may play a role as a platform to recruit host cytoplasmic proteins. For instance it could interact with host proteins that bind capped RNA, host factors that mediate the transcription–translation coupling observed in Bunyavirales22 or be involved in replication-related activities. It should however be noticed that, contrary to influenza PB2 627-domain, it is not possible to identify a non-conservative mutation of a residue in LACV-L ZBD that would differentiate one LACV strain to another and might reflect host specificity.The structure presented here reveals the organization of the entire LACV-L protein. The newly described C-terminal domains can be compared with equivalent parts of ase Fig. . LACV C-23. The present LACV-L FL structure reveals two relative positions of the endonuclease and the CBD with respect to the core, that we call “LACV-L conformation 1” and “LACV-L conformation 2” 17 and Severe Fever with Thrombocytopenia Syndrome virus L (Phenuiviridae family)25 published while this manuscript was under review  with a magnesium ion and the catalytic aspartic acids in an inactive open state conformation . Movements occurring between pre-initiation and elongation also differ between the two viral polymerases. For instance, the priming loop takes a completely different position and organization when it extrudes from the active site in the two cases . Influenza polymerase priming loop, which is 35-amino acid long, has 17 residues extruded into the solvent in a disordered loop, and projects towards the PB1–PB2 interface helical bundle12 . LACV-L priming loop, which is 20-amino acid long, orders itself at the surface of thumb ring and lid domains . Its extrusion is coupled with a Peribunyaviridae-specific unwinding of the α-helix 72 of the mid-domain into a loop, enabling an interaction between the priming loop and the extended mid-thumb ring linker  on ice in lysis buffer  with EDTA-free protease inhibitor complex. After lysate centrifugation at 48,384 × g during 45 min at 4 °C, protein from the soluble faction was precipitated using (NH4)2SO4 at 0.5 mg/ml and centrifuged at 104,630 × g for 45 min, 4 °C. Supernatant was discarded, proteins were resuspended back in the same volume of lysis buffer and centrifuged at 104,630 × g during 45 min at 4 °C. LACV-L FL was purified from the supernatant by nickel ion affinity chromatography after a wash step using 50 mM Tris–HCl pH 8, 1 M NaCl, 20 mM Imidazole, 0.5 mM TCEP, 10% glycerol and eluted using initial lysis buffer supplemented by 300 mM Imidazole. LACV-L FL fractions were pooled and dialyzed 1 h at 4 °C in heparin-loading buffer . Proteins were loaded on heparin column and eluted using 50 mM Tris–HCl pH 8, 1 M NaCl, 0.5 mM TCEP, 5% glycerol. LACV-L FL was then mixed in a 1:3 molar ratio with both 3′  and 5′  vRNA oligonucleotide ends which had been pre-annealed by heating at 95 °C for 2–5 min followed by cooling down on bench at room temperature. During overnight dialysis at 4 °C in a gel filtration buffer  LACV-L FL formed a complex with vRNA, which was ultimately resolved on the S200 size exclusion chromatography column.Sequence-optimized synthetic DNA encoding a N-terminal his-tag, a TEV protease recognition site, and the LACV-L FL  was synthetized (Geneart) and cloned into a pFastBac1 vector between NdeI and NotI restriction sites 13 as a model after removal of the endonuclease. There are two L protein complexes per asymmetric unit . The initial map after molecular replacement revealed that the core of the L protein and bound RNA were little changed but there was clear density for the endonuclease in a new position. In addition, there was extra density for the previously missing C-terminal domain. This density was improved by multi-crystal and non-crystallographic two-fold averaging using PHENIX29. Based on secondary structures predicted from an extensive multiple sequence alignment of Peribunyaviridae L proteins30, it was possible to build an approximate model of much of the C-terminal domain , except the CBD for which there is no density, including identification of the zinc-binding site co-ordinated by highly conserved cysteine and histidine residues. The two C-terminal domains from the two complexes in the asymmetric unit interact around a non-crystallographic two-fold axis in such a way that the extreme terminal helix of one packs against the C-terminal domain of the other, forming a domain swapped dimer. When the accurate structure of the C-terminal domain was obtained by cryo-EM, the X-ray model could be improved  and 5′ (9–16) vRNA was concentrated to 5 mg/ml. The 5′ (1–10) vRNA end (5′-AGUAGUGUGC-3′) was later soaked into crystals in 1:2 molar ratio. Initial hits were dense and round precipitates that appeared in 100 mM Tris pH 8.0, 100 mM NaCl, and 8% PEG 4000. Upon manual reproduction in hanging drops, they grew as thin hexagonal plates, but were soft and fragile and diffracted only to ~8 Å. To improve the resolution, crystals were soaked in a stepwise manner with increasing concentration of the glycerol cryo-protectant, reaching 30%. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), using a helical collection strategy and maximum transmission of the ID29 beamline. Crystals are of space-group –16 and 5For cryo-EM experiments, LACV-L FL in complex with pre-annealed 3′ (1–16) and 5′ (9–16) vRNA at 0.2 mg/ml was mixed with 5′ (1–10) vRNA hook in a 1:2 molar ratio. UltraAuFoil grids 300 mesh, R 1.2/1.3 were negatively glow-discharged at 30 mA for 1 min. 3.5 µl of the sample was applied on the grids and excess solution was blotted away with a Vitrobot Mark IV (FEI) , before plunge-freezing in liquid ethane. Grid screening and cryo-EM initial datasets were collected on a 200 kV Thermofischer Glacios microscope equipped with a Falcon II direct electron detector.31. Automated data collection was performed with SerialEM using a beam-tilt data collection scheme32, acquiring one image per hole from nine holes before moving the stage. Micrographs were recorded in super-resolution mode at a 165,000× magnification giving a pixel size of 0.4135 Å with defocus ranging from −0.8 to −3.5 µm. In total, 16,498 movies with 40 frames per movie were collected with a total exposure of 50 e−/Å2  coupled to a Gatan K2 direct electron detector camera33. Images were binned twice, resulting in 0.826 Å/pixel size. Further initial image processing steps were performed in cryoSPARC v2.14.234. CTF parameters were determined using “patch CTF estimation” on non-dose weighted micrographs. Realigned micrographs were then manually inspected using “Curate exposure” and low-quality micrographs were manually discarded for further image processing resulting in a curated 16,015 micrographs dataset  and subjected to 3D classification with coarse image-alignment sampling (7.5°) using a circular mask of 170 Å and 10 classes , originating from 4 out of the 10 classes from the third 3D classification  cutoff. Figures were generated using ChimeraX40.All the cryo-EM maps, namely pre-initiation map, elongation-mimicking map, CBD-mid domain map, and ZBD-mid domain map were superimposed using Chimera41 and presented with ESPript42.Six sequences of major Peribunyaviruses  were aligned using MUSCLEFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Reporting summary


Moreover, multiple studies have highlighted that cell culture in traditional Petri dishes commonly fail to predict drug sensitivity. Conversely, animal models present differences in tumor biology compared with human pathologies, explaining why promising therapies tested in animal models often fail when tested in humans. The surging complexity of patient management with the advent of cancer vaccines, immunotherapy, and precision medicine demands more robust and patient-specific tools to better inform our understanding and treatment of human cancer. Advances in stem cell biology, microfluidics, and cell culture have led to the development of sophisticated bioengineered microscale organotypic models (BMOMs) that could fill this gap. In this Perspective, we discuss the advantages and limitations of patient-specific BMOMs to improve our understanding of cancer and how these tools can help to confer insight into predicting patient response to therapy.Cancer is a leading cause of death across the world and continues to increase in incidence. Despite years of research, multiple tumors  still have limited treatment options in the clinic. Additionally, the attrition rate and cost of drug development have continued to increase. This trend is partly explained by the poor predictive power of traditional  BMOMs enable researchers to control cell organization and compartmentalization as well as the flow of nutrient and waste products,in vitro and in vivo models is illustrated in BMOMs are commonly defined as microscale Microfluidics is defined as the manipulation of fluid volumes at the submillimeter scale. At the microscale level, the effects of surface tension and capillarity dominate gravity and inertia, which makes fluid behavior highly predictable.in vitro platforms that operate in the milliliter scale. Therefore, the use of small volumes in BMOMs enables better recapitulation of physiologically relevant factor concentrations while avoiding sample dilutions or manipulations that may incur in sample loss .An often-mentioned advantage of BMOMs is their small sample requirement, which enables work with limited samples .Another advantage of BMOMs is their potential for scalability, which is desirable for basic studies and indispensable for extensive drug screening .Additionally, we envision that microfluidics, coupled with BMOMs, could be instrumental in facilitating primary sample research. The inconsistency and labor-intensive nature of sample processing are common concerns for researchers working with primary samples. Predictable microscale physics can be leveraged to optimize laboratory procedures resulting in new strategies to standardize and automate primary sample processing .in vivo models, BMOMs offer additional monitoring abilities. Most BMOMs are amenable to high-resolution microscopy, which can help investigate biological processes in detail .in vivo models are becoming more amenable to imaging techniques , they are limited by the shortcomings intrinsic to the imaging technique .in vivo systems often result in the “black box” effect. This caveat is described as the user's inability to determine the relationships between input and output. Conversely, due to the increased control that the user can exert on customizing the organotypic model, the stochastic nature of BMOMs is reduced compared to in vivo models.Compared with Monitoring capabilities could be further improved in BMOMs via the integration of miniaturized sensors .in vivo models is their modular nature which enables scalable complexity. In BMOMs, researchers can determine the number of components desired for a specific design ,in vivo models, which are top-down systems . Furthermore, BMOMs can be customized to include comprehensive and controlled environmental stimuli for mechanistic studies. Examples of environmental stimuli include mechanical cues in vivo,in vitro cultures. An example is a recent report that showed that cell proliferation in organotypic 3D models, which more accurately matched in vivo proliferation, was vastly different from the proliferation observed in traditional 2D models.in vitro.An advantage of BMOMs over In recent years, we have witnessed a handful of studies developing patient-specific organotypic models for drug-testing applications.in vivo models. Despite their similarities, mice and humans present differences at many different biological levels.A vast majority of BMOMs report using human cell lines or patient-derived cells, which often constitutes an advantage over First, studies have shown that the transcriptomic landscape of mice and humans is significantly different, thereby translating into physiological and functional differences.in vivo models will translate to human pathophysiology.Second, it is known that some diseases  cannot be modeled in mice due to the apparent mouse-to-human differences.Finally, even in those mouse models in which human cells have been implanted, the microenvironment and surrounding cells (commonly referred to as the stroma) have a mouse origin.in vivo and in vitro models with the additional benefits derived from their small scale. Harnessing physics at the microscale in BMOMs provides many advantages: allows for a high degree of control and customizability over the building components of the model and lower reagent costs compared to traditional in vitro and in vivo models. However, BMOMs also present limitations that must be taken into consideration before future perspectives of these models can be discussed.Overall, the main advantages of BMOMs are rooted in a careful balance between in vitro platforms or animal models, most of these models remain confined to engineering research and are excluded from traditional cellular and molecular biology laboratories. Adoption of these technologies has been hindered by both scientific and logistical challenges.Although BMOMs offer multiple advantages compared with traditional Traditionally, BMOMs have focused on mimicking concrete tissue structures such as the liver sinusoid, the nephron, or the lung epithelium, which are simpler systems compared with a complete organ .in vivo .There have been some attempts to connect BMOMs mimicking different organsAdditionally, this gap between BMOMs and human physiology becomes wider when we consider human behavior and behavioral responses. Notably, multiple diseases  involve higher level functions, resulting from interactions between multiple organs and kingdoms.Traditionally, soft lithography has been the most used technique to fabricate microfluidic platforms to hold BMOMs. Soft lithography relies on pouring the elastomer polydimethylsiloxane (PDMS) on top of a negative template, fabricated by UV-lithography. Next, the PDMS device is commonly bonded to a glass substrate using oxygen plasma to generate the microfluidic platform. This approach provides researchers with a relatively versatile and fast approach to prototyping. However, soft-lithography is not amenable to mass production, as compared to injection molding, one of the most common techniques used to fabricate Petri dishes and well-plates.Additionally, PDMS presents additional limitations as a material for laboratory use. PDMS is a porous material that absorbs small hydrophobic molecules, which can significantly affect cell biology. Furthermore, multiple molecules used in cell biology , as well as drugs used in the clinic , are absorbed into the PDMS, which will significantly decrease the effective concentration in culture.Further engineering limitations in BMOMs are illustrated in their limited throughput potential. BMOMs are a versatile tool to model complex scenarios, including interactions between multiple cell types and tissue structures . However, many BMOMs trade complexity for throughput potential, thereby leading to sophisticated platforms poorly suited for the parallelization and high-throughput capacity required for drug screening. Additionally, the use of specialized equipment to operate these complex models  further limits their throughput. Furthermore, most BMOMs require in-depth, hands-on training, partially explaining why they have remained limited to research environments with little translation into clinical settings. Conversely, most microfluidic platforms designed for high-throughput applications  rely on simplistic designs  unable to capture the complex structure and interactions of biological tissues and organs in BMOMs.Despite the advantages of BMOMs, their implementation in research and clinical settings remains limited.Arguably, the main barrier to a wider implementation of BMOMs is a lack of deep understanding of the critical components required for each disease model to successfully predict patient outcome. One approach that can facilitate the development of predictive BMOMs is to apply the Adverse Outcomes Pathway (AOP) developed within the field of systems toxicology. AOP defines the key events and relationships between the molecular inciting event and adverse outcome.in silico modeling of BMOMs. For example, studies are being continued to illustrate the importance of the microbiome and cross-kingdom interactions, as well as stress, diet, and environmentin silico and “wet” models to unravel their importance in a specific mechanism according to AOP. Thus, it is unlikely that we will be able to develop the ideal BMOM without the help of animal models and clinical studies. A synergy between the engineering community, biologists, computational biologists, and clinicians can allow the information obtained from human and animal studies to be incorporated into BMOMs and computational models and vice versa. Furthermore, developing predictive patient-specific BMOMs to achieve the goals of precision medicine poses even greater challenges that can only be overcome through a concerted and multidisciplinary effort in research, engineering, industry, and patient advocate foundations (The complex process of rational design for BMOMs may benefit from the integration of computational systems biology, which aims to mathematically model complex, nonlinear biological systems.ndations .97–101J.M.A., K.Y.P., and M.V. contributed equally to this work.


It was found that domain configurations change significantly with temperature. Hierarchical nanodomain structure is widely observed in domain patterns acid-etched at 25 °C, due to the orthorhombic-tetragonal phase coexistence. By contrast, the majority part of those acid-etched at − 60 °C are simply some long parallel stripes, while a small amount of banded structure appears in broad stripes inside some grains. A nearly 63° intersectional angle is seen between two adjacent sets of parallel stripes in the domain pattern of a cuboid-shaped grain, indicating that orthorhombic phase remains down to − 60 °C. The domain patterns acid-etched at 80 °C become even simpler, mainly consisting of long parallel stripes that are several hundred nanometers wide and have quite straight edges. Fundamental issues associating with the possible domain configurations and the acid-etching were discussed on the simple mathematical basis.Domain structure often greatly affects piezoelectric performance of a ferroelectric ceramic. Accordingly, a convenient method that can well characterize the domain structure at various temperatures is highly desired for understanding the underlying mechanism. An improved acid-etching technique was recently developed for such purpose. Domain structure of poled 0.96(K Currently, lead zirconate titanate and their derivatives are widely used in a variety of technological products such as ultrasonic transducers, actuators and sensors, because of the performance characters of excellent piezoelectric properties and temperature stabilities. However, due to the toxicity of lead element, demands to replace them with lead-free alternatives become increasingly strong in recent years. Accordingly, NbO3. The intrinsic part comes from relative ion shift in crystal lattice, whilst the extrinsic part arises primarily from domain-wall motion4. Particularly, the extrinsic contribution to piezoelectric coefficient d33 can take high percentages over 75% in some small-grained BaTiO3 ceramics5 and more than 70% in certain PbO3-based ceramics6.Domain structure often plays a significant role in affecting the piezoelectric performance of a ferroelectric ceramic. It is known that the piezoelectric properties can be generally resolved into intrinsic and extrinsic contributions10. Aqueous solutions of HF, HCl and HNO3 acids are often used as the etchant. The acid-etching method allows easily observing the domain patterns of a large number of grains thereby drawing a reliable conclusion on a basis of statistical analysis5. The disadvantages are that it usually cannot provide the information about the polarization orientations in the domains. The TEM observation has a very high resolution but requires very thin specimens. It is quite sensitive to the specimen preparation, during which domain structure may change irreversibly due to the stresses of mechanical polishing and focused ion-beam milling. The PFM observation is also one of the most useful methods, because of the simple specimen preparation and usually no need to operate in vacuum. It allows not only the visualization of domain structure, but also the quantitative determination of the polarization orientation by simultaneous analysis of out-of-plane and in-plane piezoresponse signals11. However, limited view window and quite time consuming seem to be the disadvantages.How to visualize thereby characterizing the domain structure, particularly catching the subtle changes in it with temperature, is believed to be significantly important for the understanding of piezoelectric performance. Morphological observation of chemically etched surface with an optical microscope or a scanning electron microscope (SEM), structural observation of thin specimens prepared by mechanical polishing and ion milling with a transmission electron microscope (TEM) and detection of surface polarization distribution with a piezoelectric force microscope (PFM), are presently the three most commonly used methods for the visualization of domain structure. Each of these observation methods has its own advantages and shortcomings. Surface chemical etching bases on the different etching rates of the opposite polarities (the positive end and the negative end) of a polarizations dipole18, PFM23 or the combination of TEM and PFM29. A few of these studies investigated the evolution of domain configurations at room temperature and above it26. The attempt of acid-etching was made quite earlier for a pure KNN ceramic30. However, most of these previous studies were conducted for unpoled KNN-based ceramics. More efforts should be paid to the domain structure of poled KNN-based ceramics, as it is known that ferroelectric ceramics show the piezoelectricity only after poling while domain configurations may change greatly upon poling. On the other hand, the acid-etching observation method is considered as a relatively reliable technique for revealing the true domain structure of a ferroelectric ceramic31. Polycrystalline grains inside the ceramic bulk are clamped three-dimensionally by their neighboring grains, whereas those at the surface experience only a half clamping from one side. Due to this clamping difference, domain structure in the grains at the surface may differ significantly from that in the inner grains. The situation may become even more severe for the case of ceramic lamellas used in the TEM observation, as the thickness is usually less than the grain sizes. The grains in these ceramic lamellas are free of clamping in the thickness direction and thereby being clamped in two dimensions only. This may lead to a substantial change in domain structure even if no additional stress is introduced during the specimen preparation, where mechanical polishing and ion milling are adopted. It is obvious that the reliability of a result is greatly dependent on the character of ceramic specimens used in the study of domain structure. Ideally, the specimen preparation that does not alter the frozen-in state is required so that the observed domain patterns can represent the frozen-in domain configurations of grains inside the ceramic31. For the reasons just described, the author’s group paid continuously their study efforts onto the domain structure of poled KNN-based ceramics by means of the acid-etching in the past few years36.Numerous studies on domain structure have been performed for the various compositional KNN-based ceramics so far, mostly by TEM3-based ceramics at low temperatures due to the lack of a proper technological way. Apparently, this is not a satisfactory situation for the comprehensive understanding about the underlying physical mechanisms of piezoelectric performance. Therefore, a feasible method that can well characterize the domain structure at different temperatures is highly needed. To meet the requirement, we recently made a preliminary attempt in our previous study to extend the acid-etching technique such that it can be used at various temperatures37. In this study, we further optimized this technique and pursued a more systematical study on domain configurations of poled 0.96(K0.48Na0.52)(Nb0.96Sb0.04)O3–0.04(Bi0.50Na0.50)ZrO3 ceramics (which possess a significantly large piezoelectric coefficient d33 of 535 pC/N) at − 60 °C, 25 °C and 80 °C, respectively. The results showed that the newly developed method is not only effective but also reliable for conveniently investigating the domain structure at different temperatures. In addition, fundamental issues relevant to the domain structure and the acid etching were discussed on the simple mathematic basis.It is noteworthy that no studies had been performed for the domain structure of NbOd33 value of 535 pC/N at room temperature and a mass density ρ of 4.548 g/cm3, which corresponds to a relative density ρ′ of approximately 98.7%36. Therefore, these KNNS-BNZ ceramics might be taken as a good representative of those high-d33 KNN-based piezoelectric ceramics obtained in recent years. Figure ε′ and ε′′, respectively) versus temperature dependences measured for the poled KNNS-BNZ ceramic at 100 kHz upon heating under a rate of 1.0 °C/min. Two peaks are seen in these curves in the temperature range below 120 °C, which correspond to the rhombohedral-orthorhombic (R-O) phase transition and orthorhombic-tetragonal (O-T) phase transition, respectively. The corresponding peak temperatures, TR-O and TO-T, are about − 40 °C and 49 °C in the ε′-temperature curve, and are approximately − 47 °C and 39 °C in the ε′′-temperature curve, respectively. Compared to those prepared under shorter sintering time35, the present KNNS-BNZ ceramics shows a slightly lower TO-T but a little larger ρ′ and a further enhanced d33. The X-ray diffraction (XRD) analysis indicated that poled KNNS-BNZ ceramics at room temperature are in O–T phase coexistence with the phase volume ratios of VO = 54.2% and VT = 45.8%, respectively36.The KNNS-BNZ ceramics used in this study show a quite large The evolution of domain structure with temperature in poled KNNS-BNZ ceramics was explored in this study through examining the domain patterns that are revealed by acid-etching at room temperature, − 60 °C and 80 °C, respectively. These three temperatures were chosen by taking both the character of phase transitions of the KNNS-BNZ ceramics and the experimental feasibility into consideration. Poled KNNS-BNZ ceramics are in the O–T phase coexistence state of slightly more orthorhombic phase at 25 °C according to the XRD analysis, and are expected from the dielectric measurement data to be mainly of rhombohedral phase at − 60 °C and tetragonal phase at 80 °C, respectively. The low etching temperature was set to − 60 °C so that the possible freezing of the acid aqueous solution can be avoided and the experiment temperature is in the operational temperature limit of the Espec SU-261 chamber. The high etching temperature was set to 80 °C from safety consideration that the danger of highly corrosive acid due to the possible evaporation might occur at further high temperatures. In addition, the optimum etching time at 80 °C was found to be about 12 s, which is quite short but still in the experimentally convenient range of precise control and good repeatability. In contrast, the optimum etching time was confirmed to be about 46 min at − 60 °C and about 4 min at 25 °C. Thus, the acid-etching rate changes greatly with temperature.35. Frequently, fine parallel strips are further recognized inside some of these bands. Most of fine parallel strips are in the shapes of short segments and intersect with the side edges of corresponding matrix bands. Part of fine parallel strips have their widths even narrower than 100 nm. Such type of domain configurations that fine and short strips exist in broad stripes correspond to the hierarchical nanodomain structure reported earlier in literature and is believed to play an important role in enhancing the piezoelectric properties39. As shown in Fig. 38. Low polarization anisotropy in the phase coexistence region results in a decrease of domain wall energy and a consequent reduction of domain widths. It is worth noting that part of band edges are in curved shapes, perhaps due to the low polarization anisotropy associating with the O–T phase coexistence at room temperature. Interestingly, irregularly shaped watermarks that correspond to 180°-domains are frequently observed in unpoled ferroelectric ceramics39 are hardly observed in the present domain patterns, indicating that the poling of KNNS-BNZ ceramics is complete. Furthermore, it is worth noting that there are also a large number of bands inside which no fine nanodomain structure can be found completely or partly. Possible mechanism resulting in this phenomenon will be discussed later.Figures TR-O values determined upon a heating process are higher than − 60 °C, but largely resembles the above described case of TO-T. Besides, as shown by the domain patterns in Fig. In contrast to those complicated domain patterns that were acid-etched at room temperature, the domain patterns acid-etched at − 60 °C are much simpler, as shown in Figs. Figures 39 are observed as alternating light and dark parallel stripes in the domain patterns. It is known that there are 8, 12 and 6 energetically equivalent orientations of spontaneous polarizations in each single phase of rhombohedral, orthorhombic and tetragonal phases, respectively. In the framework of pseudocubic denotation, the equivalent orientations of spontaneous polarizations are along the <111> c directions for rhombohedral phase, along <110> c for orthorhombic phase and along <100> c for tetragonal phase, respectively. Three types of domain walls  are permitted in rhombohedral phase. Four types of domain walls  are allowed in orthorhombic phase, while only two types of domain walls (the 90° and 180° types) are permissible in orthorhombic phase.In order to get a better understanding about the various domain patterns obtained at different temperatures, several fundamental issues concerning domain structure and acid etching will be discussed below. The characterization of domain structure with acid-etching technique is based on the different etching rates between the positive and negative ends of ferroelectric dipoles at the polished surface (the observation plane). Domains that are actually of lamellar shape in three-dimensional space31, our discussion will be limited only to the cases of uncharged domain walls below. A head-to-tail arrangement and equal projections to the normal direction are essential conditions for two adjacent polarizations of Because the uncharged domain walls are believed to be energetically more stable than those charged domain wallsThe intersection angle ϕ between the two sets of parallel planes  with their normals This is the intrinsic angle observed in a plane that is perpendicular to the two set of parallel planar domain walls. However, if the observation plane is not perpendicular to the set of parallel planar domain walls, the observed intersection angle should differ from ϕ. In general, in a third arbitrary plane with its normal direction of Possible values of intrinsic angle ϕ are calculated to be approximately 45° and 90° in rhombohedral phase, be approximately 35.3°, 45°, 65.9° and 90° in orthorhombic phase, and be approximately 45° and 90° in tetragonal phase, respectively. Accordingly, possible Since the crystallographic orientations are randomly aligned in a non-textured ceramic, the crystallographic planes exposed to the observation plane are diverse for different polycrystalline grains. Usually, it is difficult to assign the polarization directions of domains without knowing the specific crystallographic axes for a polycrystalline grain. This seems to be a shortcoming for the acid-etching observation method in comparison to the AFM and TEM observation ones. Occasionally, however, when some grains with regular shape in microstructure are seen in the observation plane, valuable information could be drawn. As shown in Fig. On the other hand, whether two adjacent domains of polarizations The larger the absolute value of entclass1pt{minimaAn improved acid-etching technique that is convenient and reliable for characterizing the domain structure at different temperatures was successfully developed in our recent study. Examination on evolution of domain configurations in poled KNNS-BNZ ceramics was performed by comparing the various domain patterns that are acid-etched at − 60 °C, 25 °C and 80 °C, respectively. The study showed that both acid-etching rate and domain configurations change significantly with temperature. Hierarchical nanodomain structure is widely observed in part of broad stripes at 25 °C, which should be the consequence of orthorhombic-tetragonal phase coexistence. In contrast to those complicated domain patterns observed at 25 °C, the acid-etched domain patterns at − 60 °C become much simpler. The latter domain patterns usually consist of simple and long parallel stripes, while only a small portion of banded structure appear in broad stripes in some grains. An intersectional angle of nearly 63° was observed between adjacent sets of parallel stripes in the domain patterns of cuboid-shaped grains, which indicates that the poled KNNS-BNZ ceramics at − 60 °C are still at least partly in orthorhombic phase. The domain patterns acid-etched at 80 °C are typically composed of parallel long stripes, mostly being several hundred nanometers wide and showing quite straighter edges. Hierarchical domain structure is only occasionally observed at this temperature. In general, the obtained results support the previous speculation that the piezoelectric properties and the piezoelectric temperature stabilities of KNNS-BNZ ceramics are greatly affected by the phase transitions and the corresponding domain structure. Some fundamental issues that are related to the domain configurations and the acid-etching were treated on the simple mathematical basis.0.48Na0.52)(Nb0.96Sb0.04)O3–0.04(Bi0.50Na0.50)ZrO3 (hereinafter denoted as KNNS-BNZ) ceramics were prepared via the conventional solid-state reaction route as per previous studies36. Starting raw materials of reagent-pure carbonate and oxide powders were precisely weighed in desired stoichiometric ratio and ball-milled in nylon jars with alcohol as medium. After drying, the obtained mixture was calcined at 890 °C for 8 h. Followed was a second round of ball milling and drying. The resultant ceramic powder was then granulated and pressed at 300 MPa into small thin disks. Lastly, the small thin disks were sintered by a two-step temperature profile in ambience. In this sintering process, the temperature was first quickly raised 1170 °C, kept for 5 min, then cooled down in 5 min to 1070 °C and maintained for 20 h before subsequent natural cooling.The 0.96 in the volume ratio of 1:1. The polished ceramic specimens and the polyethylene container filled with the mixed acid aqueous solution were together put into the temperature-controlled chamber. After the temperature got stabilized, the specimens were dipped into the mixed acid solution and etched in it for desired time lengths. Subsequently, the acid-etched specimens were taken out from the acid solution, washed with deionized water, and were then dehydrated by tissue paper. The etching time was varied according to the etching rate at different temperatures. Specifically, the optimized etching time conditions were 46 min at − 60 °C, 4 min at 25 °C and 12 s at 80 °C, respectively. Domain patterns were finally studied on a scanning electron microscopy (SEM) JSM-7610F.