Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.The online version contains supplementary material available at 10.1007/s00239-022-10071-3. Coagulation (blood clotting) is the process of gel formation of the blood at injured and/or damaged tissue sites. Under normal conditions, blood circulates through arteries, capillaries and veins but responds to tissue damage by clot formation. Therefore, it is essential to have a rapid and effective coagulation mechanism to prevent blood loss as well as undesirable coagulation within veins (thrombosis). Clotting is regulated by a complex cascade which involves more than two dozen proteins including serine proteases (Doolittle ).The cascade is divided into three pathways: the intrinsic pathway, the extrinsic pathway and the common pathway (Grover and Mackman ). The inMore than half of the proteins which belong to the blood coagulation cascade are serine proteases which are highly similar to each other in terms of function and structure: They serve as activators/deactivators of downstream proteins with their protease activity and possess the same or similar domain arrangements (Doolittle ). It is Gla – EGF – Fxa inhibition – Trypsin. Similarly, cofactors of the system, FV and FVIII, also share similar domain arrangements: Cu-oxidase (4–6 repeats) – F5 F8 type C. Finally, Plasma Kallikrein and Factor XI (FXI) also share the same domain organization: PAN 1 (× 4) – Trypsin. Gene duplications, fusions, fissions and terminal losses can lead to novel domain arrangements . Therefand Albà ).Analogous to blood coagulation in vertebrates, Arthropods have a hemolymph clotting system to prevent hemolymph loss and provide immunity . The evolution of blood coagulation cascade proteins is widely studied Patthy , 1990; D; D1990; https://zivgitlab.uni-muenster.de/domain-world/dw-helper) was used with settings ‘-r “gene[: =] \\s*([\\S] +)[\\s]*”’ for proteomes from Ensembl and the settings ‘-r “GN[: =] \\s*([\\S] +)[\\s]*”’ for proteomes from Uniprot. NCBI proteomes could not be handled with that program, so a python script (provided together with the study data) has been used. To detect domain arrangements, we used PfamScan , NCBI ) and Uninn et al ) using Pnn et al ). The blWe used OrthoFinder (v2.3.12) to detect orthologous proteins between species (Emms and Kelly ). We perOne of the goals of this study was to identify potential origins of domains involved in the blood coagulation cascade. Specific databases were created containing only sequences from our data set belonging to one specific domain. To shed light on the domain evolution of the blood coagulation cascade, domain level reciprocal BLASTP (v2.9.0)  searcheIf the query domain is found within the first three hits  of the reciprocal search, the target is considered a good hit. To perform a BLASTP search, we first built a local database using all respective domains from a target proteome.In a few cases, orthology analysis was not be able to identify some proteins in the studied species which should be present according to the current literature and in some cases orthology analysis identify some proteins which should not be present in the studied species. In both cases the same methodology was used: We used the Ensembl genome browser  to deteMUSCLE (v5.1) was used to align blood coagulation cascade proteins in the proteomes (Edgar ). We buiOrigin of Domains and Evolution of Domain Arrangements).Putative orthologs of blood coagulation proteins were searched in the study species using OrthoFinder and BLASTP. Table Tursiops truncatus. However, according to Exonerate results, the genomic region in T. truncates that spans most of the FXII protein has four nonsense mutations . Exonerate runs on other cetaceans, namely sperm whale (Physeter catodon) and blue whale , showed similar or worse alignments. Therefore, we confirmed that FXII is pseudogenized in cetaceans. According to previous studies, a pseudogene conversion by point mutations has led to this protein prediction . FXII iba et al ).PAN 1) followed by a Trypsin protease domain (Supplementary Table S2). Both PK and FXI are not present in jawless vertebrates, cartilaginous fish and ray-finned fish confirmed by exonerate, synteny and BLAST results. The last ancestral protein of PK and FXI should have appeared before the divergence of lobe-finned fish. It is found that, while PK is present in almost all tetrapods except for the cetaceans, a PK duplication before the divergence of mammals led to the emergence of FXI as discussed by Ponczek et al  are paralogs that have the same domain arrangement: four tandem apple domains . Both, FXI and PK have the domain arrangement PAN 1 – PAN 1 – PAN 1 – PAN 1 – Trypsin. As for FXII, the domain arrangement is fn2 – EGF – fn1 – EGF – Kringle – Trypsin. Plasminogen has the domain arrangement PAN 1 – Kringle – Kringle – Kringle – Kringle – Kringle – Trypsin. Lastly, the domain arrangement of Prothrombin is Gla – Kringle – Kringle – Thrombin light – Trypsin. Even though serine proteases of the cascade are well conserved, cofactors are diverse in terms of the number of repetitions of the domains. FV had a variable number of LSPR domain repeats among different vertebrates, ranging from 24 in species Balaenoptera musculus to 38 in species Physeter catodon, while FVIII has a different number of domain repeats in Copper oxidases (Supplementary Table S3–4).In terms of their domain arrangements, most of the blood coagulation cascade proteins are well conserved in all vertebrates. FIX, FX, Protein C, and Protein Z have identical domain arrangements , a non-insect Arthropod, was investigated as its coagulation proteins are widely studied. Hemolymph clotting in L. polyphemus is controlled by a proteolytic cascade similar to vertebrates. The cascade is controlled by coagulation factors B, C, and G, Proclotting enzyme, Coagulogen, Transglutaminase, and Coagulation inhibitors .The hemolymph coagulation cascade of ka et al ; Theopolka et al ; Schmid ka et al ). Even tDrosophila melanogaster as a reference species. Hemolymph clotting in D. melanogaster is controlled by a small set of proteins: Fondue, Hemolectin, Transglutaminases, and Prophenoloxidases  , Transgen et al ; Dushay en et al ). Therefen et al . Similaren et al ). With hD. melanogaster hemolectin and von Willebrand factor share three domains: VWD, TIL and C8. Therefore, we built a phylogenetic tree of the proteins which have VWD, TIL and C8 domains with shared domains and similar modularity, it is highly possible that these two proteins share a common ancestry. The constructed phylogenetic tree with bootstrap values can be found in Fig. Besides the identical domain arrangements of coagulation FXIII subunit A and Transglutaminase, Gla – EGF – Fxa inhibition – Trypsin), while FVII has Gla – EGF – Trypsin. Besides the above arrangements, Gla—EGF—Fxa inhibition arrangement is also found within the different domain arrangement of Protein S (Gla – EGF – Fxa inhibition – EGF CA – EGF CA – Laminin G 1 – Laminin G 2). To reveal evolutionary histories of these domain arrangements, we performed reciprocal BLASTP searches using samples of FVII and FX from the most basal species. Unfortunately, reciprocal BLASTP searches on the domain level did not result in reciprocal matches in most of the cases. Next, we extended our search including the best three hits of the BLASTP results. BLASTP searches using Petromyzon marinus and Eptatretus burgeri FVII and FX domains against Oikopleura dioica, Ciona intestinalis, and Stylophora pistillata yielded two proteins in O. dioica: E4WZI2 and E4X518  are likely the result of gene duplications. In humans, FVII, FX, and Protein Z are located close to each other on the forward strand of 13th chromosome (2–9 kbp between each of them) which supports the role of gene duplication in the emergence of these serine proteases.Protein S and FVII  only differ in their terminal domains. While FVII has serine protease activity provided by its terminal Gla, Kringle and Trypsin, respectively , our most basal species with FXII, and the lobe-finned fish . Two hits were found for Kringle and Trypsin domains, ENSLACP00000015011 (tissue type plasminogen activator) and ENSLACP00000010869 (urokinase plasminogen activator), respectively. It is proposed that FXII arose from duplication of Hepatocyte Growth Factor Activator (HGFAC) , Danio rerio, Callorhinchus milii, and Homo sapiens as queries against Petromyzon marinus and Eptatretus burgeri proteomes showed that there is not a reciprocally “perfect match” between FV and any jawless vertebrate protein. Hephaestin has a reciprocal match in the lamprey proteome which is also the best hit for FV. Therefore, this lamprey protein is most likely an ortholog of Hephaestin instead of FV. To further support this finding, we built a gene tree using all possible putative orthologs of FV found by BLASTP (71 candidates for E. burgeri and 31 candidates for P. marinus), together with H. sapiens, D. rerio, and C. milii FV, FVIII, hephaestin, and ceruloplasmin. While FV and FVIII from shark, zebrafish and human are clustered together, hephaestins and other jawless vertebrate proteins are found in different clades . . Lastly, we performed protein to genome Exonerate runs using human FV against lamprey and hagfish genomes. Even though there is no good alignment that spans most of the FV protein, there are short sequences in jawless vertebrates that align to smaller fragments of FV (see study data). Searches on the current genomic data of jawless vertebrates, do not yield a concrete result. Considering all these analyses, FV is therefore potentially not present in jawless vertebrates but rather emerged in Gnathostomata after the split from Cyclostomata around ∼600 mya. However, since the available jawless vertebrate genomes are in low quality, this finding could be an artifact of the fragmented genomes.As for FV and FVIII, it was suggested that a single copy of FV is present and FVIII missing in jawless vertebrates (Doolittle ). Howevedes Fig. . BesidesTIL, VWD, and C8 containing proteins. The phylogenetic tree of TIL, VWD, and C8 domains-containing proteins from human, mouse, zebrafish, horseshoe crab, and fruitfly shows homology between vertebrate von Willebrand factor and Arthropod hemolectin. However, to gain a deeper understanding of the evolution of von Willebrand factor and Hemolectin, future studies should include animals deeper in the phylogeny and fungi species, Fusarium sp. and Aspergillus sp., as their proteomes contain more ancestral versions of the aforementioned domains.As for von Willebrand factor, tracing the domain arrangement evolution of the protein is more difficult due to its modular structure. Similar to FV and FVIII, von Willebrand factor is also composed of a highly modular domain arrangement, making it similar to Arthropod Hemolectin . In all studied animal groups, there are similar modular Unfortunately, due to the long evolutionary time frame of over ∼600 mya and low sequence similarity, it is difficult to determine possible evolutionary trajectories of blood coagulation cascade protein domains. However, our work here increases the resolution of Doolittle’s work on vertebrate blood coagulation cascade evolution (Doolittle ).The blood coagulation cascade is very complex and, although some proteins are missing in early vertebrates, the coagulation proteins are structurally well conserved among all vertebrates. While Arthropod hemolymph clotting seems functionally similar to the blood coagulation cascade, only two proteins in the blood coagulation cascade (FXIII subunit A and von Willebrand factor (vWF)) have similar domain arrangements to hemolymph clotting proteins Transglutaminase and Hemolectin, respectively. The remainder of the blood coagulation proteins, including all the key proteins, are found to have different evolutionary histories compared to Arthropod hemolymph clotting proteins.Here, not only do we show new data on the emergence of FXIII, Protein C and Protein Z, but we also propose possible evolutionary trajectories for vertebrate coagulation factors which were derived from domain similarity assessments. The first appearance of FV could not be exactly defined. Although a previous publication  showed Supplementary file1 (PDF 313 kb)Below is the link to the electronic supplementary material.


In this study, calcium carbonate nanoparticles (CCNPs) and calcium oxide nanoparticles (CONPs) are synthesized by the carbonization/calcination of calcium oleate. CONPs are an essential inorganic material, and they are used as catalysts and as effective chemisorbents for toxic gases. CCNPs are widely used in plastics, printing ink, and medicines. Here, calcium oleate is used as a starting material for the preparation of CCNPs and CONPs. This calcium oleate is prepared from calcium hydroxide and oleic acid in ethanol under mild reflux conditions. The effect of the calcination temperature of calcium oleate is examined during the synthesis of CCNPs and CONPs. By simple carbonization/calcination, calcite-type CCNPs and CONPs are prepared at <550 °C and >600 °C, respectively. The synthesized nanomaterials are analyzed by various physicochemical characterization techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy, thermogravimetric analysis (TGA) with derivative thermogravimetry (DTG), and scanning electron microscopy (SEM) with energy dispersive X-ray analysis. An X-ray diffractometer and the Scherrer formula are used to analyze the crystalline phase and crystallite size of prepared nanoparticles. TGA techniques confirm the thermal stability of the calcium oleate, CCNPs, and CONPs. The SEM analysis illustrates the dispersive behavior and cubic/spherical morphologies of CCNPs/CONPs. Furthermore, the obtained results are compared to the CCNP and CONP samples prepared using calcium hydroxide. As a result, the carbonization/calcination of calcium oleate produces monodispersed CONPs, which are then compared to the CONPs from calcium hydroxide. Additionally, from calcium oleate, CONPs can be prepared on a large scale in a cheap, convenient way, using simple equipment which can be applied in various applications. However, the technique causes the considerable agglomeration of particles during synthesis, leading to a bimodal size distribution [Calcium carbonate ). The calcination process determines the structural characteristics of CaO, which is an active sorbent for CO2 [Calcium oxide (CaO) is applied in various fields because it is inexpensive, non-corrosive, and easy to handle with excellent basicity compared to typical homogeneous base catalysts, such as catalysts, bactericides, additives in refractories, flue gas desulfurization, pollutant emission control, and particularly critical adsorbents for toxic chemical agents ,12. Aparecursors ,14,15. C for CO2 . CaO nan for CO2 .(1)CaO, Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) with derivative thermogravimetry (DTG), and scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDAX).Calcium hydroxide (99.9%), ethanol (99.8%), and oleic acid (90%) were purchased from Aldrich Chemicals, and they were used as received without any further purification.Scheme 1 clearly illustrates the formation of calcium oleate by a mild reflux condition and the preparation of CCNPs/CONPs from calcium oleate via the simple carbonization/calcination (pyrolysis) method. On the other hand, CCNPs/CONPs were prepared using calcium hydroxide. For this, an appropriate amount of calcium hydroxide was taken alone into a silica crucible, and it was calcined for 5 h in a muffle furnace at different temperatures (450–800 °C) under static air conditions. The formation of CCNPs and CONPs from calcium hydroxide via the simple calcination method is shown in Scheme S1.An appropriate amount of calcium hydroxide (1 mol) and oleic acid (2.5 mol) was added to ethanol. The reaction mixture was refluxed for 5 h, and the obtained solid material  was filtered and washed several times with ethanol. Subsequently, the calcium oleate was dried and then carbonized/calcined at various temperatures for the production of CCNPs and CONPs. In a typical growth experiment, an appropriate amount of calcium oleate was taken into a silica crucible, and it was carbonized/calcined for 5 h in a muffle furnace at different temperatures (450–800 °C) under static air conditions. The muffle furnace was cooled to room temperature, and then the final product (white color powder) was collected from the crucible. Figure 1. The patterns exhibited an intense diffraction peak around 29.4° (2θ) due to (104) diffraction and low intense peaks around 23.1, 31.4, 36.0, 39.4, 43.2, 47.1, 47.4, 48.5, 56.6, 57.5, 60.7 and 64.8° (2θ) attributed to the (012), (006), (110), (113), (202), (024), (018), (116), (211), (112), (122) and (220) planes, respectively, confirming the formation of CCNPs with the calcite polymorph phase [The XRD patterns of CCNPs and CONPs are shown in ) due to 4 diffracph phase . The obt (024), 0, (116), ph phase ,20. The Figure 2. As seen in the spectra of the CCNPs, three strong bands centered at around 713, 875, and 1450 cm−1, corresponding to framework vibrations of ν4, ν3, and ν2, respectively, and were considered to arise from CO32− [4, ν3, and ν2), ν2 vibration was strong and broad due to the crystalline structure of CONPs and CCNPs without major defects [−1 may be attributed to the stretching vibration of −OH groups of physically adsorbed or lattice water molecules [–1. The intensities of the ν4, ν3, and ν2 vibrations decreased for CONPs prepared at 600 and 625 °C, which might be due to the transformation of CCNPs into CONPs. In addition, a sharp band was observed at 3645 cm–1 and attributed to the asymmetric –OH stretching vibration [−1, corresponding to C−H asymmetric and symmetric vibrations in the methyl groups, respectively, which were not observed in the CCNPs and CONPs. The small peak at 1705 cm−1 is assigned to the −C=O stretching vibration of carboxylate groups in the calcium oleate [−1 were due to asymmetric stretching bands for uni- and bidentate carboxylate groups associated with calcium ions [FTIR spectra of synthesized calcium oleate, CCNPs, and CONPs were recorded to determine their existing surface functionalities. The infrared spectra of the calcium oleate, CCNPs, and CONPs are shown in om CO32− . Among t defects . The weaolecules ,24,25. Dibration . This asm oleate . The intium ions . The hydium ions .2. After that, no weight loss was observed up to 1000 °C, suggesting the stability of the CONPs. This result matches well with the XRD result. The thermal decomposition of the synthesized CCNPs and CONPs was evaluated at temperatures ranging from 25 to 1000 °C using TG/DTG analysis . An initial minute quantity of weight loss below 150 °C was due to the desorption of physically adsorbed water molecules. The second/final weight loss due to the decomposition of the occluded organic template occurred between 600 and 770 °C. CCNPs were decomposed and transferred into CONPs and CO2 (g). The loss of around 47% of the initial mass in this temperature range was due to the escape of CO2 from the system after the decomposition of CCNPs. Furthermore, we observed the absence of decomposition up to 1000 °C from the thermogram, which is credited to the purity of the synthesized calcite nanoparticles. Two separate weight losses were displayed in the CONPs prepared at above 600 °C. The first weight loss was observed from 380 to 450 °C, and the second/final weight loss was observed from 550 to 770 °C for the CONPs prepared at 600 °C. The CONPs prepared above 650 °C showed the weight losses from 350 to 420 °C and 500 to 670 °C. The first weight loss was due to the removal of hydroxyl groups on the surface of CONPs, and the final weight loss was due to the incomplete conversion of CCNPs into CONPs during calcination at a temperature beyond 600 °C [The thermal stability of synthesized calcium oleate was examined by TGA, and the corresponding results are shown in d 600 °C . The derFigure 4, Figure 5 and Figure 6 show the SEM images of CCNPs and CONPs synthesized at different temperatures. The experiments were carried out to study the effect of reaction temperature on the morphology of CCNPs and CONPs fabricated from calcium oleate via a simple carbonization/calcination method. The nanoparticles obtained at 600 and 650 °C were nearly spherical and uniform in size, but particles formed at 450–550 and 650–800 °C were cubic-shaped and aggregated. The nanoparticles formed at 650 °C had unique properties such as good dispersion and narrow size distribution. The diameters of the nanoparticles were in the range of about 60–80 nm. The average diameter of the CONPs was estimated as 70 nm. This result is consistent with the size calculated by the Debye–Scherrer equation in XRD studies, which implies a single crystalline nature of prepared nanoparticles. The EDAX spectra of CCNPs and CONPs were recorded for the samples synthesized from calcium oleate at different carbonization/calcination temperatures . The corresponding spectra displayed the appearance of carbon (C), oxygen (O), and calcium (Ca) elements , and the corresponding SEM images are shown in CCNPs and CONPs were synthesized by the simple carbonization/calcination of calcium oleate and prepared by the interaction of calcium hydroxide and oleic acid. Below 600 °C, aggregated CCNPs with calcite polymorph phase were obtained. However, sphere-shaped CONPs were obtained by the calcination at 600–650 °C, and the CONPs were well dispersed with narrow size distribution. The diameters of CONPs were virtually the same, and the range in diameter was calculated as 60–80 nm. The spherical shape of CONPs was turned into different morphologies beyond 650 °C, with moderate aggregation. Additionally, the CONPs’ particle size increased in the temperature range from 700 to 800 °C. Hence, the monodisperse CONPs were obtained from calcium oleate at optimal calcination temperature (650 °C). On the other hand, CCNPs and CONPs, as prepared from calcium hydroxide, showed polydispersed and aggregated particles. Thus, this study provides the carbonization/calcination route of calcium oleate, a versatile and simple technique that can be used for the large-scale production of CCNPs and monodispersed CONPs that can be applied in various vital and industrial applications, including papers, paints, plastics, and biomedical products.


Bacillus thuringiensis (Bt) and other bacteria are pesticidal pore-forming toxins. Since 2010, when the ABC transporter C2 (ABCC2) was identified as a Cry1Ac protein resistant gene, our understanding of the mode of action of Cry protein has progressed substantially. ABCC2 mediates high Cry1A toxicity because of its high activity for helping pore formation. With the discovery of ABCC2, the classical killing model based on pore formation and osmotic lysis became nearly conclusive. Nevertheless, we are still far from a complete understanding of how Cry proteins form pores in the cell membrane through interactions with their host gut membrane proteins, known as receptors. Why does ABCC2 mediate pore formation with high efficiency unlike other Cry1A-binding proteins? Is the “prepore” formation indispensable for pore formation? What is the mechanism underlying the synergism between ABCC2 and the 12-cadherin domain protein? We examine potential mechanisms of pore formation via receptor interactions in this paper by merging findings from prior studies on the Cry mode of action before and after the discovery of ABC transporters as Cry protein receptors. We also attempt to explain Cry toxicity using Cry–receptor binding affinities, which successfully predicts actual Cry toxicity toward cultured cells coexpressing ABC transporters and cadherin.Cry proteins from  Bacillus thuringiensis (Bt), a pathogen of insects and nematodes. Other bacteria such as Paenibacillus popilliae and Paraclostridium bifermentans produce several Cry proteins [Cry protein is one of the classification groups of pesticidal proteins from bacterial pathogens . The pesproteins ,4. Each proteins ). Bt andm/kDa) is produced as a parasporal body during sporulation. In the gut of hosts, proprotein is solubilized and processed into its activated form (~60 m/kDa) by gut digestive fluids. The three-dimensional structure of activated Cry protein comprises three highly conserved domains. Domain I, the N-terminus domain, is involved in pore formation and has a colicin-like fold with seven alpha helices. The middle domain, Domain II, has a beta-prism structure and is in charge of receptor interaction and pesticidal specificity. The beta-sandwich structure of Domain III, the C-terminus domain, is critical for receptor interaction and structural stability. Cry genes are encoded on plasmids and appear to have been diversified through domain swapping via homologous recombination to adapt to their host and expand their host range [Cry proteins are classified as α-pore-forming toxins whose alpha helices form pores on the cell membrane. Cry proprotein  was identified to be a responsible gene for high resistance to Cry1Ac protein in the n et al. . In thate target . Since te target ,33 for de target  because e target . The dise target ,35 basede target . Lack ofe target . HoweverHelicoverpa armigera [Plutella xylostella [B. mori [H. armigera and Chrysomela tremula [Aside from ABCC2, researchers discovered that other insect ABC transporters could function as Cry protein receptors. ABCC3, a possible paralog of ABCC2 in lepidopteran species, also functions as a Cry1A receptor ,37,38, barmigera , Cry1Ac lostella ,41, and [B. mori , while s tremula ,44. The Therefore, the conventional understanding of Cry protein’s mode of action has progressed over the last decade. Nevertheless, the process of pore formation via receptor interactions is still poorly understood. We compare ABCC2 and cadherin to explore what occurs on the cell membrane from receptor interaction to pore formation and the role of receptors mediating Cry toxicity and present a current molecular model for Cry protein pore formation via receptor interactions. We also aim to explain Cry toxicity in binding kinetics, including synergism between ABCC2 and cadherin.First, why do Cry proteins require receptors to exhibit toxicities? Activated Cry proteins can form pores on receptor-free artificial lipid bilayers . NeverthB. mori ABCC2, Spodoptera exigua ABCC3, and Tribolium castaneum ABCC4A [ABC transporters appear to be a shared receptor for Cry proteins. Aside from Cry1–ABCC2/C3, Cry2–ABCA2, and Cry3–ABCB1, other Cry subfamily and ABC transporter combinations may be discovered. Cry8Ca, generally known as a coleopteran-killing protein, for example, exerts its toxicity through m ABCC4A ,37. ThisPectinophora gossypiella with cadherin null mutation [Spodoptera frugiperda and B. mori did not affect Cry1F susceptibility [H. armigera caused Cry1Ac resistance but no significant change in Cry2Ab susceptibility [Diabrotica virgifera [Leptinotarsa decemlineata [S. frugiperda did not affect Cry1Ab susceptibility [Trichoplusia ni does not bind to Cry1Ac [Conversely, cadherin seems to be an uncommon receptor across the Cry protein family. The AZP-R strain of mutation  is resismutation . Cadheritibility ,48. CRIStibility . Cadheriirgifera  and Cry3mlineata . Cadherio Cry1Ac . These eB. mori cadherin (BmCad). Cry1Aa binding to BmABCC2 and cadherin is affected by the deletion of loop 3 (441–444 aa) and a double cysteine mutant R281C-I369C, in which the cavity between domain I and domain II loop 2 is impaired. These areas are close to the boundary between domains I and II, including the Asp222-Arg281 salt bridge, one of the four domain I–II salt bridges. They may serve as the switch triggering partial unfolding of domain I and oligomerization. R366 and R368 are responsible for binding to BmABCC2 and are located near the Arg234-Glu274 salt bridge. Thus, the ABCC2-binding site on Cry1A is close to the two domain I–II salt bridges. Meanwhile, the BmCad-binding site contains domain II loops 1–3, and all three loops contribute equally to binding affinity.Using the data from , Figure ABC) is defined as binding affinity/ABC-mediated toxicity . Hence, ABC-mediated Cry toxicity can be calculated as binding affinity/pore formation efficiency.Does the pore formation-helping activity of ABC transporters correlate with binding affinity? 2 = 0.9019, p < 0.0001)  A. Other  0.0001) A. These  so much B. Partic PEABCC2 B, suggesM. sexta , likely Given the low and frequently undetectable nature of cadherin-mediated toxicity, there has been a notable lack of data on cadherin-mediated toxicity and binding affinity in the same species or among Cry families. There are examples of Cry1Aa mutants combined with BmCad–TBR . The BmCad-binding affinities of Cry1Aa domain II loop mutants, which included six mutants with ~10–50-fold higher binding affinity than wild-type Cry1Aa, did not correlate with BmCad-mediated toxicities ,55,56.It is plausible that insect susceptibility to Cry protein is basically based on the sum of contributions of each receptor and synergism between receptors to pore formation, although many other factors  are involved in determining Cry susceptibility. As ABCC2 exhibits remarkable pore formation-inducing activity for Cry1A proteins, it is expected to be the primary susceptibility determinant in general A. When lAltogether, Cry resistance caused by a cadherin mutation indicates the existence of a synergism between cadherin and ABC transporter(s) which is the primary determinant of susceptibility to the Cry protein. In contrast, having no high resistance which is caused by a mutation in an ABC transporter does not necessarily mean that the ABC transporter is not involved in Cry susceptibility. When multiple ABC transporters make the almost same contributions, Cry susceptibility is maintained at almost the same level by remaining receptor(s) even if one of them is lacking.D = ~100 nM) in comparison to ABCC2 and cadherin (KD = ~0.1–1 nM) and does not induce Cry1A oligomerization [Elucidating the role of APN and ALP has been one of the important issues in studies of Cry’s mode of action. According to our definition of Cry receptors, APN and ALP cannot be considered as a Cry receptor at this stage in general. They bind to Cry1A protein domain III with lower affinity  expression in Sf21 cells conferred ~30% cell death after 5-h incubation with 3.75 µg of activated Cry1Ac protein [H. armigera [S. exigua and APN1 and APN2 in Aedes aegypti did not affect Cry1Ac, Cry1Ca, and Cry1Fa susceptibilities or Cry4Ba and Cry11Aa susceptibilities, respectively [Drosophila melanogaster larvae expressing Manduca sexta APN1 (MsAPN1) were susceptible to Cry1Ac [Drosophila larvae expressing P. xylostella ABCC2 exhibited ~100-fold higher Cry1Ac susceptibility than larvae expressing MsAPN1 in a dose-dependent manner [M. sexta will help validate the role of MsAPN1.One example that APN confers Cry susceptibility was reported in  protein . However protein . Taking armigera ,63, it sectively ,65. Droso Cry1Ac , having t manner . Use of How about the role of APN as a “secondary receptor” in the sequential binding model? APN has been believed to receive Cry1A oligomers from cadherin and insert them into the cell membrane. If this is the case, APN should be essential for Cry toxicity. Nevertheless, the significant Cry1A resistance generated by ABCC2 mutations in many lepidopteran insects ,63,68,69P. xylostella is confusing. First, different results of CRISPR-Cas9-mediated PxABCC2 and PxABCC3 knockouts were reported. Liu et al. and Zhao et al. reported that single knockouts of either PxABCC2 and PxABCC3 caused no or several-fold Cry1Ac resistance but the double knockout of them caused >8000-fold resistance [Molecular understanding of Cry1Ac susceptibility in sistance ,41, suggsistance ,42. ThusP. xylostella APN1 and APN3a were functional receptors for Cry1Ac [P. xylostella. However, the roles of the two APNs cannot be determined until the mechanisms underlying many putative “synergisms” are unraveled. In contrast, the results from Guo et al. suggest that different mechanisms based on putative unknown “synergisms” underlie the primary Cry1Ac susceptibility determinant in the same species. They reported that single knockouts of PxABCC2 and PxABCC3 independently caused 413- and 724-fold Cry1Ac resistance , suggestr Cry1Ac . Heterolr Cry1Ac . This suHow does Cry oligomerization occur? Before oligomerization, each monomer must bind to a receptor to expose the helices essential for oligomerization and pore formation, whether in solution or in the cell membrane. Since the Cry1A–cadherin interaction has a 1:1 stoichiometry  and the How does a Cry–receptor complex assemble? Interactions of domain I helices between conformationally changed monomers induce oligomerization. The probability and efficiency of oligomerization are expected to depend on the density of receptor molecule(s) and diffusion speed on the basis of membrane fluidity.It has been debated whether oligomerization or membrane insertion occurs first in the Cry mode of action. However, because these two actions occur concomitantly in most α-pore-forming toxins , this arWe, likely, do not need to consider the role of prepores in the case of ABCC2-mediated pore formation. It is fair to assume that the domain I helices of a conformation-changed monomer binding to ABCC2 are inserted into the cell membrane. The completion of the assembly of four Cry–ABCC2 complexes directly signifies pore formation . ConversWhy does ABCC2 efficiently mediate pore formation of Cry1A proteins? The apparent difference in molecular characteristics between ABC transporters such as ABCC2 and other Cry-binding proteins is the transport activity of substrates. This activity derives energy from ATP hydrolysis and the accompanying conformational change between open and close states, which might underlie the pore formation mechanism via ABCC2. Heckel hypothesized that inserting a Cry prepore into the outward-facing cavity of open state ABCC2 aids in the pore formation in the lipid bilayer . MoreoveTM-resistant S. exigua strain, which lacks a part of the nucleotide-binding domain (NBD) II in the C-terminal, serves as a functional receptor for Cry1A proteins [These hypotheses were appealing, but they seem implausible for several of the reasons listed below. First, the introduction of Cry protein helices into the ABCC2 cavity appears physically impossible. Cry1A protein possesses complex cavities in domain II that bind to ABCC2 . The Cryproteins . These mM. sexta brush border membrane vesicles (BBMV) containing lipid rafts [H. virescens and M. sexta BBMV [What, then, are the underlying factors? One possibility is the distance from the cell membrane. Since the extracellular region of ABCC2 only comprises a very small part of the protein, the Cry1A–ABCC2 interaction occurs very close to the cell membrane , which cid rafts . Some stxta BBMV  and the xta BBMV . Althougxta BBMV . TherefoB. mori [D/EC50) is more than 90-fold lower via BmCad than via BmABCC2. In terms of distance from the cell membrane, the Cry–cadherin interaction occurs relatively far from the cell membrane when compared with the Cry–ABCC2 interaction because of the length of cadherin repeat(s) and membrane–proximal extracellular domain (MPED)  . CadheriD for Cry1A is ~0.5 nM, and hardly ever dissociate from cadherin. Furthermore, because their Cry1Aa-binding sites overlap [The molecular mechanism of ABCC2 and cadherin synergism in assisting Cry1A pore formation remains unknown. The essence of the synergism is that ABCC2 can mediate pore formation using abundant Cry1A proteins binding to cadherin. Some researchers believe that cadherin’s sequential binding partner is ABCC2 rather than APN, and that ABCC2 is responsible for receiving tetrameric prepores from cadherin and inserting them into the cell membrane ,76,88. N overlap , cadheriThe synergism could be explained by the interactions between Cry1A–ABCC2 and Cry1A–cadherin complexes via domain I helices rather than sequential binding via domain II. When four Cry–cadherin complexes form a tetramer, ABCC2 finds it difficult to insert it into the cell membrane. However, it is possible in the case of a single Cry1A–cadherin complex (monomer) and assemblies of two to three Cry1A–cadherin complexes (dimer and trimer). Interactions between partial-unfolded domain I of Cry receptor complexes, which is the same force that causes Cry oligomerization, may draw domain I helices of cadherin-binding Cry1A protein into the cell membrane, resulting in pores comprising four Cry1A–ABCC2/Cry1A–cadherin complexes . The ratD ABC and KD cad, 50% of the receptors are occupied by the Cry protein. Thus, the amount of binding Cry protein at the EC50 toward ABC-expressing cells (EC50 ABC) is required for killing 50% of cells via ABC transporters.The magnitude of the synergistic effect varies across ABC-cadherin combinations and across Cry proteins. For instance, the BmABCC2-BmCad synergism causes 10-, 1000-, and 100-fold higher toxicity of Cry1Aa, Cry1Ab, and Cry1Ac when compared to BmABCC2 alone, and no synergism was observed in Cry1Fa ,42. WhatD cad is comparable with or lower than KD ABC, the binding amount of Cry protein required for killing 50% cells is achieved at a lower concentration (EC50 ABC/cad) when compared with EC50 ABC . Toxicities predicted using this formula are strongly correlated with the actual synergism-mediated toxicity observed in in vitro assays   , indicatIn this context, a synergism between ABC transporters is normally unlikely, as Cry binding affinities correlates with ABC-mediate toxicity , i.e., pBasically, the killing mechanism of Cry protein appears to be quite simple: the more pores that are generated, the greater the toxicity. We present a model of Cry1A pore formation via receptor interactions. By inducing a conformational change in the Cry protein, ABCC2 mediates membrane insertion of Cry domain I helices. Cry–ABCC2 complexes diffuse on the basis of membrane fluidity, and collisions between Cry–ABCC2 complexes generate dimers, trimers, and tetramers via intradomain I helices interactions, according to the kinetic model for the stepwise Cry oligomerization process . The CryD ABC/PEABC. The synergism (ABC/Cad)-mediated toxicity strongly correlates with KD ABC * KD Cad/PEABC (= EC50 ABC * KD Cad). In the future, in vivo toxicity toward pest individuals may be predicted if a much more accurate mathematical model is built using our formulas as prototypes by incorporating contributions to other receptors (if exist), the expression levels of receptors, and other factors filling gaps between in vivo and in vitro.The Cry toxicity can be explained by binding affinity to its receptor. The ABCC2-mediated toxicity is represented as KPore formation mechanisms are a major gap in our understanding of Cry protein mode of action. Three-dimensional structures of Cry pores and Cry receptor complexes are required for a complete understanding of the process, from receptor interactions to pore formation, and to test the hypothesized model we propose here. It is also critical to understand how Cry1A domain II loops contribute to pore formation.Based on solid mechanistic and kinetic logic, we will be able to design Cry protein variants active against Cry-resistant strains and naturally nonsusceptible pests. There are two examples of successful Cry protein engineering that overcame Cry resistance. First, Cry1Mod proteins, which lack domain I alpha-1 and spontaneously form oligomers, are effective against a wide range of Cry1A-resistant lepidopteran strains, including ABCC2- and cadherin-deficient strains ,91. OverT. ni cadherin and overcoming resistance [Escherichia coli expression system that is not suitable for the expression of 12-transmembrane helix transporters. An alternative method for Cry engineering toward ABC transporters is required for freely designing Cry toxicity and its specificity.The other example is phage-assisted continuous evolution (PACE), which allowed Cry1Ac protein affinity maturation to sistance . PACE is


This article presents a study of changes in the microstructure of Hadfield steel depending on the tensile deformation and cold rolling with the strain/stress level. It has been established that the change in the “σ-ε” curve (at ε = 5%) is accompanied by a 1.5-times decrease in the strain-hardening coefficient. At ε = 0 to 5%, the structure contains dislocation loops, the interweaving of elongated dislocations, single-layer stacking faults. At ε = 5%, the structure contains multilayer stacking faults and mechanical microtwins. At ε > 5%, there is an intense microtwinning with no long dislocations and stacking faults. The most intense twinning develops in the range of deformation degrees of 5–20%, while the number of twins in the pack increases from 3–4 at ε = 10% to 6–8 at ε = 20%. When mechanical twinning is included, a cellular dislocation substructure begins to develop intensively. The cell size decreases from 700 nm at ε = 5% to 150 nm at ε = 40%. Twinning develops predominantly in systems with the largest Schmid factor and facilitates the dislocation glide. The results may be of interest to the researchers of the deformation processes of austenitic alloys. The austenitic high-manganese Hadfield steel (1.2% carbon and 12% manganese) occupies a special place among wear-resistant high-carbon manganese alloys. Due to high toughness, good flexibility, and wear resistance, Hadfield steel is widely used in various industrial applications  . In the A distinctive feature of Hadfield steel is its pronounced ability to strain harden. The main strengthening mechanism of Hadfield during plastic deformation is the pile-up dislocations (dislocation arrangements may develop highly compact tangles with an increase in the degree of deformation) and the formation of twins . The cryIn ,15, an aIn the study of the dislocation structure of polycrystals of austenitic stainless steels, it was previously established that stacking faults are observed simultaneously with planar pile-up dislocations ,15,16,17Bracke et al.  showed tNikulina et al.  showed tTo increase the ductility of TWIP steel, Zhi et al.  introducKim et al.  used in-In the previous study , the patAt present, the role of dislocation and twin substructures in the strain-hardening behavior is generally well modeled and studied, a complete analysis of the experimental data and a generalization of modern concepts in the field of deformation behavior of face-centered cubic alloys of g-Fe with a high manganese content was performed by De Cooman et al. . There aThe role of dislocations is an important parameter to be studied not only for Hadfield steel but for other alloys too. For example, the role of dislocations and related boundaries on the hydrogen embrittlement behavior of a cold-rolled 7xxx series Al alloy was studied in . A cold-However, experimental characteristics of the evolution of the deformation substructure, as well as a detailed quantification of their respective roles in the strain hardening of Hadfield steel during tensile deformation, are still rarely reported in the literature.Therefore, this study aims to establish the structural changes in Hadfield steel as a result of mechanical stress by tension and rolling, to evaluate the quantitative parameters of its dislocation substructures, and to evaluate the influence of the deformation mechanism (sliding and twinning) and the substructures formed during deformation on the strain hardening of this steel. The experimental facts and theoretical considerations of this article contribute to the current picture of deformation processes in TWIP steels, in which deformation twinning plays an important role.Austenitic Hadfield steel  was used as the study material. The elemental composition of steel is given in 3) was carried out at a temperature of 1050 °C for 30 min in a laboratory tubular electric furnace SUOL-0.4.4/12-M2-U4.2  in a vacuum. The temperature in the furnace was measured and controlled by a precision temperature controller VRT-2 using two thermocouples of the TPP 1378 type . The accuracy of temperature control and maintenance was ±0.5 °C, and the measurement error at the temperature of the upper measurement limit of 1300 °C was no more than ±3 °C, i.e., the total temperature measurement error in this furnace did not exceed ±4 °C. The state of the samples was fixed by quenching them in water at room temperature.All ingots were homogenized and normalized. Thermal heating of steel samples  with measurement of the yield stress σ (MPa). The stress exceeding the yield stress was determined by the ratio of the load to the actual cross-sectional area of the sample with uniform deformation at a rate of 0.2·10−3 s−1. The tension axis was oriented along the rolling direction. Based on the results of the experiments, graphs of the dependences of the flow stress σ (MPa) on the degree of plastic deformation ε (%) were plotted and the strain hardening coefficients Θ (Θ = dσ/dε) for the Hadfield steel were calculated.Mechanical tests were also carried out at room temperature on the samples with dimensions of 0.5 × 15 × 75 mm2. Sample foils were examined for narrow twins and dislocation structures in a JEM-2100  analytical transmission electron microscope operated at 200 kV. JEM-2100 was equipped with a STEM system and an energy-dispersive micro(nano) system Inca Energy TEM 350 Analysis . The method of arbitrary secant lines was used to determine the volume ratio of dislocation substructure and twins by TEM images. At least five TEM images for each type of sample with the minimum of 15 arbitrary secant lines were analyzed.For TEM analysis, the samples were prepared by mechanical grinding and electrolytic thinning in a mixture of hydrogen peroxide in phosphoric acid at a temperature of 80 °C and twin jet electropolishing. The electropolishing agent was a supersaturated solution of chromic anhydride in phosphoric acid at a temperature of 60 °C and with an electric current density of 0.5–0.7 A/cmSamples for optic microscopy using Neophot-21  were prepared by mechanical and electrolytic polishing and etching as detailed in .v) of the dislocation substructure (DSS) formed during deformation, we used the planimetric method described in [The volume fractions of deformation microtwins were determined by the planimetric method . To deteribed in ,26,27 foribed in .b is the Burgers vector of dislocations; ∂φ/∂l is the gradient of the curvature of the foil or the curvature-torsion of the crystal lattice χ. The value χ = ∂φ/∂l was determined by shifting the extinction contour (Δl) at a controlled angle of inclination of the foil (Δφ) in the microscope column using a goniometer.To study the DSS evolution with the strain/stress level, we measured such parameters as the density of dislocations ,25 and tThe results of tensile tests of Hadfield steel samples are presented by the tensile stress-strain (σ − ε) curve as shown in −3 s−1, there are two stages of plastic flow development II1 and II2 with different strain hardening coefficients (Θ(II1) = 3·103 MPa and Θ(II2)= 2.1·103 MPa, respectively). The inflection on the σ − ε curve corresponds to the value of the degree of tensile deformation ε = 0.05 (5%) and, presumably, corresponds to the moment of inclusion in the twinning deformation. The shape of the curve in Upon deformation of Hadfield steel at the constant tensile rate of 0.2·10Before deformation, the steel microstructure is represented by grains with an average grain size of 150 μm without carbides at the boundaries and without slip bands inside the grains a. Upon rThe steel structure before deformation is homogeneous austenitic and the results of the EDX analysis of the distribution of elements in the selected direction  do not sTEM results showed that at the initial degrees of deformation (up to ε = 5%), the substructure of the material is represented by randomly distributed dislocations and individual mechanical microtwins .The presence of regions with a predominance of long dislocations elongated along one or two <110> directions is typical for tensile deformation with ε < 5% a. These Upon reaching ε = 5%, multilayer stacking faults and mechanical microtwins appear in the structure a. At tenThus, at the initial tensile deformation degrees within the initial grains, the formation of a dislocation substructure begins. With an increase in the degree of plastic deformation, the density of dislocations at the subboundaries increases, and the crystallographic misorientations between subgrains increase, which leads to a sharp change in the hardening coefficient when the degree of deformation reaches 5%, as shown in In previous studies ,30,31, tv) and twinning (Pvtw) (g (Pvtw) a, as welg (Pvtw) b on the The results shown in As seen in Thus, a correlation is observed between changes in the substructure, the activation of new deformation mechanisms , and stages of plastic flow. With an increase in the deformation degree, the development of twinning in several sliding systems and the formation of packets of microtwins is observed. The number of twins in the packets increases with increasing deformation, that is, the TWIP effect is presumably observed, accompanied by a decrease in the hardening coefficient by about 1.5 times.In order to reveal the relationship between the stages of deformation and the quantitative parameters of the dislocation substructure, the following parameters were measured: scalar and excess density of dislocations and the density of tensile deformation twins. The dependence of the quantitative parameters of the substructure  on the plastic deformation degree is shown in 13 m−2, at the deformation with a degree of 40%, at which the destruction of most samples occurs under tension. Under tension, the parabolic growth of the scalar dislocation density ρ continues up to ε = 30%. At higher degrees of deformation, the accumulation of dislocations saturates, corresponding to values of 1.4·1013 m−2. Under tension, the destruction of the sample occurs before saturation of the dislocation density can be reached, and the growth rate of the scalar dislocation density is much lower than during rolling. The excess dislocation density grows along a similar curve with an increase in the degree of deformation by tension and rolling  it reaches 5·10−8 m−2 at ε = 90% (1 and II2.The density of microtwins during tensile deformation increases up to 4·10 ε = 90% b. Accordtw) and dislocations (εdisl) to the deformation was determined under the assumption of the additivity of their contribution:The moment of inclusion of twinning in the deformation coincides with an inflection on the flow curve leading to a decrease in the strain-hardening coefficient, i.e., twinning plays a significant role in the deformation of steel. In this regard, the contribution of microtwins  It is important to note that the TEM analysis of the structure of Hadfield steel did not reveal martensite anywhere, all the observed reflections of diffraction patterns are reflections of the γ-phase c. The stSince the density of dislocations in areas containing microtwins is lower, sliding through twins is easier than in the matrix, which is confirmed by the curves plotted based on the degree of plastic deformation of the scalar density of dislocations in grains containing and not containing microtwins .γ plane. In this case, the condition of parallelism of the planes is fulfilled: (130)γ//(102)tw and directions: [γ//[tw. The orientation of the tension axis in this area of the sample is [γ. The TEM images To refine the geometry of twinning, microtwins were investigated in individual grains of steel samples deformed both by tension and rolling with ε = 25% .As can be seen from The analysis of twinning geometry in specific grains showed that twinning develops mainly in systems with the highest Schmid factor a. HoweveThe results obtained indicate the important role of the slip plane orientation in newly formed microtwins and the Schmid factors for twinning. These results are also consistent with the assumption that the slip mechanism is facilitated by increasing the number of twinning systems and are in agreement with the theory of the twinning-induced plasticity effect, as described in ,16,35,40The change in the course of the flow curve “σ − ε” (at ε = 5%) was accompanied by a sharp (1.5 times) decrease in the strain hardening coefficient and corresponded to the inclusion of the twinning mechanism in the deformation of steel.At the deformation degree from 0 to 5%, the structure contains dislocation loops, the interweaving of elongated dislocations, single-layer stacking faults, and a cellular dislocation substructure. At deformation ε = 5%, the structure contains multilayer stacking faults and mechanical microtwins. At ε > 5%, there is an intense microtwinning in different systems, with no long dislocations and stacking faults in the material.The most intense twinning with the formation of packets of twins develops in the range of deformation degrees of 5–20%, while the number of twins in the pack increases from 3–4 at ε = 10% to 6–8 at ε = 20%. Thus, there is a clear correlation between changes in the substructure of the Hadfield steel, the activation of new deformation (twinning) mechanisms, and the stages of plastic flow.When mechanical twinning is included in deformation, a cellular dislocation substructure begins to develop intensively. The cell size decreases nonlinearly from 700 nm at ε = 5% to 150 nm at ε = 40%. It is found that the dependence of the scalar dislocation density on the degree of deformation up to 30% is parabolic; at higher degrees of deformation, the accumulation of dislocations becomes saturated.The calculated Schmid factors for operating sliding systems in the matrix and twin showed that mechanical microtwinning entails orientation softening and facilitates the sliding process, while orientation hardening occurs at strains less than 5%. The results indicate the important role of the crystallographic texture in the mechanism of strain hardening of Hadfield steel and may be of interest to a wide range of researchers of the deformation processes of austenitic alloys. Knowledge of the features of work hardening of Hadfield steel can be used to improve the manufacture and processing of parts from it.


E. coli 50S subunits. A wide range of complexes with large variations in the extent of the ordered 23S rRNA and the occupancy of r-proteins were resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed an ordered density for the particle base around the exit tunnel, with domain V and the 3′-half of domain IV disordered. In addition, smaller core particles were discovered, where either domain II or IV was unfolded. Our data support a multi-pathway in vitro disassembly process, similar but reverse to assembly. Dependencies between complex tertiary RNA structures and RNA-protein interactions were observed, where protein extensions dissociated before the globular domains. We observed the formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structures and prevent non-native interactions also after folding.Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro assembly into functional 50S subunits. Here, we used cryo-EM to determine the structures of such LiCl core particles derived from  The ribosome is a large macromolecular complex translating mRNA into protein in all domains of life. Structural biology has elucidated the architecture of ribosomes, where each subunit consists of structured ribosomal RNA (rRNA) stabilized by ribosomal proteins (r-proteins) that use positively charged loops and tails linked to globular domains to stabilize the tertiary structure of the rRNA. The intricate assembly of ribosomes is, in all organisms, a tightly regulated process that has been fine-tuned by evolution. E. coli has been extensively studied in vitro, and methods were early developed to reconstitute active subunits from components -adenosyl-L-methionine  was followed by the addition of 40 pmol RlmF, or buffer (negative control) to a final volume of 30 μL. All reactions were incubated at 37 °C for up to 30 min, quenched in 2 mL of ice-cold 10% trichloroacetic acid, and incubated on ice for 10 min. The precipitated reactions were applied to BA85 nitrocellulose filters  under vacuum and washed five times with 7 mL of cold 10% trichloroacetic acid. The washed filters were placed in vials containing 5 mL of Filter Safe scintillation cocktail , shaken for 30 min, and counted in an LC6500 scintillation counter . For calculation of the fraction of modified particles, the CPM per pmol of doped SAM mix was measured. This allowed conversion of CPM per pmol of ribosomal particles to pmol of modified particles.Assays were performed in duplicates. Each reaction contained 30 pmol 50S-LiCl particles in reaction buffer 2, 0.5 mM EDTA-Tris, 6 mM β-mercaptoethanol, 0.5 mM sinefungin , 0.5 U/mL of RiboLock RNase inhibitor ) was incubated with 200 μL of Ni-Sepharose  for 30 min at 25 °C under gentle agitation in a spin-filter and, subsequently, centrifuged at 0.5 RFC for 3 min. The Ni-Sepharose beads were washed twice by incubation for 10 min in 500 µL of imaging buffer with 0.2 mM sinefungin, 0.1 U/mL of RiboLock RNase inhibitor and 30 mM imidazole, and the ribosome-RlmF complex was eluted in 200 uL of imaging buffer with 0.2 mM sinefungin, 0.1 U/mL of RiboLock RNase inhibitor and 300 mM imidazole. The eluate was sedimented in 50-μL aliquots through 50 µL of 50% sucrose cushion in imaging buffer . The pellets were resuspended in 10 μL of imaging buffer and stored on ice before being flash-frozen on cryo-EM grids. The protein content was verified by SDS-PAGE and LC-MS/MS analysis of the lane cut from the SDS-PAGE gel. Sample preparation is summarized in A 300-µL reaction containing 5.2 nmol of C-terminally His-tagged RlmF and 1.2 nmol of 50S LiCl core particles in imaging buffer . The search criteria for protein identification were set to at least two matching peptides of 95% confidence level per protein.For the LC-MS/MS analysis, the sample was digested by trypsin and the peptides were subjected to reverse phase separation on a C18-column with a 90-min gradient and electrosprayed to a Q Exactive Orbitrap mass spectrometer . Tandem mass spectrometry was performed by high-energy collision dissociation fragmentation. The data were analyzed using Proteome Discoverer 1.4 (Thermo Fisher Scientific) using the Sequest algorithm against a database of the −/pixel/second during 0.773 s for a total dose of 41.0 e−/Å2, and the sample stage was set to either 0° (3052 movies) or 15° (2196 movies) tilt. In the second session, movies were collected with 68.4 e−/pixel/second during 0.770 s for a total dose of 42.2 e−/Å2, and the sample stage was set to a 30° tilt (6120 movies). Data collection parameters are summarized in The cryo-EM specimens were prepared on QuantiFoil R 2/2 grids with 2-nm continuous carbon . The grids were glow-discharged for 40 s at 20 mA and 0.38 mBar using an EasiGlow . Plunge-freezing was done using a Vitrobot mark IV  using 3 µL of sample at 160 nM ribosome concentration after 30 s of incubation on the grid at 100% humidity and 10 °C and blotting for 3.5 s. Data were collected on two different occasions on the same Titan Krios microscope from two different grids from the same batch. The microscope was equipped with a Falcon-III direct electron detector used in integrating mode. The nominal magnification was 75,000×, corresponding to a pixel size of 1.09 Å in the specimen plane, and the acceleration tension was 300 kV. On the first occasion, movies were collected with 66.2 e2). The efficiency of the orientation distribution (Eod) parameter was calculated using cryoEF [od values in the range of 0.6–0.8  was rig ID 4YBB . The pix ID 4YBB  with ref ID 4YBB  and inte ID 4YBB . In the  ID 4YBB . Model rThe atomic model of a mature ribosome (PDB ID 4YBB), as well as a model refined against the consensus reconstruction, were rigid body fitted to each class reconstruction using UCSF Chimera 1.14 , and theThe occupancy was calculated for each structural element (chain or 23S rRNA helix) as the fraction of atoms with a map value above a threshold of 0.05. The threshold was confirmed by manual inspection of the maps. The values were normalized according to , i.e., bManual inspection to gauge occupancies of r-protein was performed by identifying the map threshold where the strongest recognizable feature could be seen. Density for 23S rRNA helices was classified as being near-native, slightly distorted, highly distorted, or disordered, which were translated into numerical values 0.1, 0.085, 0.045, and 0, respectively, for clustering and plotting purposes.E. coli secondary structure map downloaded from the Ribosome Gallery on 22 September 2020 , which differs slightly from the original publication. RNA helices not named in the map were given the number of the helix immediately preceding with an additional letter or the next number if available, cf. The boundaries for the 23S rRNA helices were taken from , specifihttps://www.scipy.org/, accessed on 10 August 2020), both for classes (columns) as well as features (rows). R-proteins that could not be identified in any of the classes were excluded from the analysis. The clustering of features was rather sensitive to the chosen parameters , but the general trends were reproducible.Hierarchical clustering using the complete linkage method and the Euclidean metric was calculated using the function scipy.cluster.hierarchy.linkage in the SciPy library   b. The fo5′) . Several5′) . In addi5′) b.Classes 2, 5, and 6 show strong similarities to in vitro reconstitution intermediates  and to i5′ and parts of domain V are folded. The large particles are mature-like, but parts of sub-domain IV3′ and parts of domain V are unfolded. The clustering of structural features , as a low-resolution protruding loop at low map thresholds . One armA number of proteins appear to be present across all classes , and in most cases also uL13 and uL24 . All of Fifteen proteins do not have clear density in any of the classes , although diffuse density prevented unambiguous assessment of some proteins. Trace levels of uL1, uL5, uL6, bL9, uL11, uL15, and bL31 were detected by LC-MS/MS , suggestThe high solubility of the CP-associated r-proteins uL5, uL18, and bL25 destabilizes the CP in the LiCl-washed ribosomes. The CP and the 5S rRNA are only discernable in the largest particle classes, 6 and 5-8 , togetheFor the three largest r-proteins in the consensus reconstruction , the long loops and tails that in the native particle interact with the 23S rRNA are invisible . The intBased on the clustering analysis, structural consequences of the removal of certain r-proteins and of unfolding of rRNA were analyzed by pair-wise comparison between the particle classes. This revealed folding and stability dependencies that are mostly additive .5′ (particularly strong density for H63) (5′ in other classes coincides with the presence of uL2 (see below). Class 1-2 has additional density for the core of domain II, a more mature-like domain 0 and H25 of domain I, but less density for domain IV and the distal half of domain III , III, VI, 0  and IVfor H63) . This is IIItail ).tail can stabilize sub-domain IV5′ through contacts between H57 and H58 in domain III and H63. The same correlation between the folding of sub-domains IV5′ and IIItail was observed under bL17 depletion [tail is mostly folded in all of the particles, independently of domain IV and uL2, suggesting that it stabilizes domain IV rRNA at the early stages of ribosome biogenesis.Based on these structures, we hypothesize that in lieu of uL2, sub-domain IIIepletion , and domepletion . Sub-dom5′ (tail and helices in domains 0 and VI) is shifted 20 Å away from the subunit interface compared to the native particle, whereas in class 2-2, this lobe is instead in near-native position.In class 2 particles, the RNA structure is stabilized by additional r-proteins. Unlike in class 1, there is no density for sub-domain IV5′ . In clasThe exit tunnel is expanded in the smaller particles, in particular in class 2-2 c,d. Thistail is closer to its native position, stabilized by H63 and possibly by the higher occupancy of uL3 and bL17. Class 3-1 is rather similar to 2-1 but H63 is more pronounced and there is more diffuse density in the H33–H35a region. Correlated to this, domain IIIIn class 3-2, domain II is in a more native position compared to 3-1. It is the smallest particle with unambiguous density for H34–H35 , correlaClass 5-1 shares attributes with classes 2-2 and 3-2, but H63 is close-to-natively folded . SimilarHigher occupancy of uL3 in 5-1 than in 3-2 seems to cause stronger density for domains 0 and VI (except H96). The same trend is also observed between 2-2 and 2-1. In the mature particle, the 128–153 loop of r-protein uL3 directly interacts with several of these helices , support5′ and more mature-like H33–H35a. The differences in domains II and IV seem correlated with increased occupancy of uL2, also seen in classes 5-2, 5-3, 5-5, and 5-6 and in the clustering analysis , placing it in a different path in Class 5-5 shows less distinct density for domain V compared to 5-4, although H79 seems ordered. Instead, the density for uL2 and sub-domain IVIn class 5-6, all the elements that are visible in either of the smaller particles in class 5 are ordered . Class 5-7 shows stronger and more well-defined density for domain V than the smaller particles in class 5. There is clear density for helices H74, H75, H76, H79, and H88, albeit shifted from their native positions . Similar3′  and H35, in a region of tertiary interactions between domains 0, II, III, IV, and V that is stabilized by the positively charged extensions of r-proteins uL2, uL22, and bL34 .Helix H49a is a stem-loop in domain III that bridges to domain II, close to the nascent-chain exit tunnel c,d. In tHelix H35, a stem-loop in domain II, packs outside H49a, contacting domains 0 (H73), IV (H64 and H65), and V (H93) g. H35 isCore particles of the LSU, where the most loosely attached ribosomal proteins and the 5S RNA have been removed using high-salt wash protocols, were first used in studies of ribosome assembly more than half a century ago ,39. HereThe removal of loosely associated r-proteins with a high-salt wash allows access to the smallest stable cores of the large subunit. These particles show density for only 4–8 r-proteins, but additional ones are possibly bound to disordered rRNA regions. With the exception of the smallest particles, the reconstructed cores show strong similarity to intermediates isolated from bL17-deficient cells or from in vitro reconstitution  and theiThe r-protein occupancy of our LiCl core particles largely agrees with previous characterizations ,6,20,42.The wide range of LSU sub-particles that we observe allows analysis of the consequences of removal of specific r-proteins. For some r-proteins, e.g., uL3, a primary binder, and uL2, a later binder  there isTo what extent does ribosome disassembly in vitro reverse the process of ribosome assembly in vivo or in vitro? Apart from the CP-region proteins uL5, uL15, and uL18, the r-proteins that bind first during assembly are also the ones that stay bound during high-salt wash. The disassembly process is sterically constrained by outer assembly layers of the ribosome, and thereby more similar to in vitro assembly where the structure forms starting from the core and continuing outwards. During the co-transcriptional in vivo process, there is an additional directionality, where at a given time only part of the rRNA can be available for folding and tertiary interactions (reviewed in ). HoweveThe reconstructed series of particles subjected to r-protein dissociation corroborates the multi-pathway of assembly ,44,45 byCompared to the well-studied early, co-transcriptional 30S assembly process , little In conclusion, the in vitro-generated 50S LiCl core particles represent a collection of intrinsically stable or meta-stable complexes, from which we can learn about the inherent structural properties of ribonucleoprotein particles, and specifically about states that are likely to occur during 50S ribosome assembly. Recent developments in the classification and analysis of heterogeneous particle populations ,50 will


Escherichia coli and for a mutant, FhuAΔ5-160, lacking the N-terminal cork domain. Urea-induced unfolding was monitored by fluorescence spectroscopy to determine the effective free energies TM ~ 49 °C in LDAO micelles to TM ~ 75 °C in lipid bilayers. Enthalpies TM and Extraction of integral membrane proteins into detergents for structural and functional studies often leads to a strong loss in protein stability. The impact of the lipid bilayer on the thermodynamic stability of an integral membrane protein in comparison to its solubilized form in detergent was examined and compared for FhuA from  This solubilization often leads to partial inactivation/inhibition of TMPs, see e.g., refs.  from Triton-X-100 micelles. Hong and Tamm established conditions and reported free energies for the reversible folding of the 8-stranded β-barrel outer membrane protein A (OmpA) in phospholipid bilayers of small unilamellar vesicles (SUVs)  is a two domain TMP, comprised an N-terminal 160-residue domain termed “cork domain” located inside a 554-residue transmembrane domain that forms a 22-stranded β-barrel in the OM of E. coli  carrying plasmid pBk7H  column coupled to an automated FPLC . Each variant of FhuA (either wt-FhuA or FhuAΔ5-160) bound selectively to Ni2+-NTA agarose and a single symmetrical peak containing purified FhuA eluted at around 200 mM imidazole in a linear imidazole gradient from 5 to 500 mM. The fractions containing purified FhuA were pooled and concentrated by ultrafiltration. Imidazole was removed by dialysis against 2 L of sodium borate buffer  containing 2 mM EDTA and 0.1% LDAO detergent and at 6.5 °C. SDS-PAGE analyses of purified FhuA variants showed single bands at 78 kDa (wt-FhuA) and at 62 kDa (FhuAΔ5-160).HisPhospholipids  were dissolved in chloroform and mixed at the desired ratios. Dry lipid films were prepared under a stream of nitrogen, followed by desiccation for at least 3 h. Lipid films were dispersed in HEPES buffer  followed by addition of FhuA solubilized in LDAO, diluting LDAO below its CMC. Sodium cholate was added to a final concentration of 0.2%. The samples were incubated for 3 h at room temperature. Cholate and LDAO were subsequently removed by 9 h of dialysis against 500 mL of HEPES buffer at 6.5 °C. The dialysis buffer was exchanged every 3 h. Finally, the molar lipid/FhuA ratio was determined as described , either solubilized in LDAO-detergent micelles at an 800-fold molar excess of LDAO or reconstituted into lipid bilayers, was mixed with buffer containing 10 M urea to obtain various final concentrations of urea ranging from 0 to 8 M in a total volume of 1 mL. Urea is a strong denaturant often used in studies on folding and stability of proteins, including To investigate heat-induced unfolding of wt-FhuA or FhuAΔ5-160, samples were prepared at LDAO/FhuA or phospholipid/FhuA ratios of 800 mol/mol and at an FhuA concentration of 1.9 μM in a total volume of 1 mL buffer. The samples were pre-equilibrated for 24 h at 25 °C. To examine heat-induced unfolding, the temperature was adjusted to 35 °C, followed by an additional equilibration for 3 h before the first fluorescence spectrum was recorded. Subsequently, the temperature was increased by 2 °C followed by an incubation of 20 min, before a new spectrum was recorded. These three steps were repeated until a temperature of 85 °C was reached.Fluorescence spectra were recorded on a SPEX-Fluorolog-322 spectrometer with double monochromators in the excitation and emission pathways. Tryptophan fluorescence was excited at a wavelength of 290 nm. The excitation and emission bandwidths of the monochromators were 2 nm and 3.7 nm, respectively. Spectra were scanned from 300 to 420 nm; the integration time was 0.05 s; and the increment was 0.5 nm.Far-UV CD measurements were performed at 30˚C on a Jasco 720 CD spectrometer using a thermostated cuvette (0.1 mm light path). Five scans were accumulated for each spectrum. The scan range was 190 to 250 nm, the response time was 8 s, the bandwidth was 1 nm, and the scan speed was 5 nm/min. The concentration of FhuA was 40 μM. The ratios of LDAO/FhuA or phospholipid/FhuA were 800 mol/mol. Background spectra of samples without FhuA but of otherwise identical composition were subtracted.XF and XU are the mole fractions of the folded and unfolded forms, respectively, T is the temperature in Kelvin, R the universal gas constant, and K the equilibrium constant. Unfolding of membrane proteins like FhuA may be monitored by systematic alteration of experimental parameters leading to unfolding, e.g., as a function of the concentration of a chemical denaturant in solution or as a function of the temperature. For unfolding monitored at increasing concentrations of a chemical denaturant like urea, the effective free energy of unfolding per mol of protein depends linearly on the concentration of the urea in the transition region of unfolding. The free energy in the absence of urea can, therefore, be estimated by extrapolation to 0 M urea, see e.g., refs. , and curea and extrapolated to curea = 0 M, respectively, and at the other selected experimental standard conditions, e.g., T, pH, concentration of buffer, selected environment , and m is a measure of cooperativity of unfolding. To obtain XF at different concentrations of urea, Eqs. Proteins may unfold in one or more steps, which may depend on the number and the stabilities of their domains. Unfolding steps may be treated individually for each domain when they are well resolved and distinguishable from another. For proteins or domains that unfold in one step, a two-state equilibrium may be described. Folded (F) and unfolded (U) states of each domain may coexist at equilibrium in transition regions. The change in free energy per mol protein that unfolds, mentclass2pt{minimfλ as the fluorescence intensity at wavelength λ, the intensity-weighted average fluorescence emission maximum Concentrations or mole fractions of folded and unfolded forms can be determined by spectroscopy when the spectroscopic signal is a linear combination of the signals of folded and unfolded forms that are proportional to their concentrations. For fluorescence emission spectra of a protein with K  at wavelength λ is a linear combination of the fluorescence signals fλ(F) and fλ(U) of the folded and unfolded forms, respectively, at wavelength λ present in the sample at molar fractions XF and XU, respectively. Defining XF:can be used to relate the fluorescence of a mixture of folded and unfolded protein to the equilibrium constant XF, the equilibrium constant of unfolding K = (1–XF) / XF is obtained:From withrom Eqs.  to8\documF and mU are the slopes of the linear dependencies of curea. Equations (Intensity-weighted average emission maxima of fluorophores mentclass2pt{minimquations  can be cquations :9\documem that describes the change in the free energy as a function of the denaturant concentration (curea), were free fit parameters. The ratio Equation G°(T) = ΔH° – TΔS° and can be obtained from the unfolding transition at the protein melting temperature TM, i.e., at the midpoint of the thermal unfolding transition, cf.  is T and Δcp is the change in heat capacity of the protein upon unfolding. TM is the unfolding temperature and In Eq. , ΔG°(T) E. coli into micelles of the detergent LDAO in aqueous buffer. We first examined unfolding of wt-FhuA and FhuAΔ5-160 by Trp fluorescence spectroscopy, which is a sensitive tool to monitor conformational changes of proteins, as the fluorescence intensity and the wavelength of the maximum of the spectrum depend directly on the molecular environment of the fluorescent tryptophan side chains, which is different for folded and unfolded forms. Unfolding of FhuA was induced at increased urea concentrations. The spectra recorded for wt-FhuA  strongly decreases the stability of FhuA. The midpoints of unfolding were determined to 6 M urea for wt-FhuA (○) and to 2.2 M urea for FhuAΔ5-160 (□). To analyze unfolding of FhuA from LDAO micelles in buffer, the intensity-weighted average fluorescence emission maxima ated Eq.  and plotated Eq.  and alsoG, which is zero at equilibrium. The linear dependence of curea  spectra  at TM ~ 69 °C that is ~ 17 °C above the TM ~ 52 °C of FhuAΔ5-160. This confirmed the strong stabilization of FhuA by its cork domain, previously observed for the urea-induced unfolding processes of the two forms.The data shown in Fig. K) vs. 1/T were linear  of both variants of FhuA for their inserted and folded forms in lipid bilayers. A TM of ~ 70 °C was obtained for FhuAΔ5-160, reconstituted into bilayers of DOPC and DOPG at a molar ratio of 9:1, and a TM ~ 85 to 90 °C was estimated for wt-FhuA after reconstitution into bilayers of DOPC and DOPG at a molar ratio of 2:3. The stabilities of wt-FhuA and FhuAΔ5-160 cannot be directly compared as the compositions of the lipid bilayers are different. However, unfolding of either wt-FhuA or FhuAΔ5-150 from LDAO micelles may be used as a common basis to examine stabilization of wt-FhuA and FhuAΔ5-150 by lipid bilayers. The midpoints of unfolding of wt-FhuA and FhuAΔ5-160 in lipid bilayers were both observed at temperatures that were ΔTM ~ 18 to 21 °C higher than observed for the corresponding LDAO-micelle-solubilized wt-FhuA and FhuAΔ5-160. The large ΔTM indicated the stabilities of both, wt-FhuA and FhuAΔ5-160 were strongly increased regardless of the lipid composition of the bilayer, reflecting strong differences between the physical properties of micelles and bilayers. For direct comparisons of the stabilities of wt-FhuA and FhuAΔ5-160, each reconstituted in bilayers of DOPC/DOPG at a ratio of 2:3 and for comparisons of the stabilities of FhuAΔ5-160 in bilayers composed of DOPC and DOPG either at a ratio of 9:1 or at a ratio of 2:3, see below.From many studies on the functions of TMPs, it is known that detergent-micelle extraction of membrane proteins often leads to their inactivation , 5.1 M (40 °C), 4.3 M (45 °C), and 1.4 M (55 °C) urea. Comparisons with unfolding experiments of FhuAΔ5-160 in micelles of lyso-lauroylphosphatidylcholine or octadecanoyldimethylammonium-N-oxide confirmed that the lipid bilayer has a stabilizing effect on FhuAΔ5-160 when compared to detergent micelles (data not shown).β-barrel domain then dominates overall fluorescence properties, which were similar to those of folded FhuAΔ5-160. In contrast to detergent solubilized wt-FhuA, the lipid membrane stabilized the β-barrel domain to an extent that the cork domain can unfold without a concurrent denaturation of the β-barrel domain . It would also explain why the cork domain greatly stabilizes the β-barrel domain against unfolding by either urea or by heat when FhuA is solubilized in LDAO micelles.Inspection of the crystal structures of FhuA  that is very similar to the stabilization observed here for wt-FhuA in lipid bilayers, which did not completely denature below 90 °C   Figs.  and 9. U160 Fig. . The OM β-barrel membrane proteins like OmpA or OmPlA. As the OMPs from E. coli examined to date have in common that their isoelectric points are all in the range between pI ~ 5.0 and pI ~ 5.9 (Kleinschmidt, β-barrel OmpA has a high activation energy of unfolding (Pocanschi et al., β-strands, the smaller β-barrels like OmpA are relatively compact, leading to a lower probability of local destabilizations of the β-barrel than for the larger β-barrels. In denaturant induced unfolding of FhuA, limited accessibility of the smaller lumen of β-barrels like OmpA to solvent or denaturant could be another reason for increased resistance against unfolding.


Natural fiber-reinforced composite (NFRC) filaments for 3D printing were fabricated using polylactic acid (PLA) reinforced with 1–5 wt% henequen flour comprising particles with sizes between 90–250 μm. The flour was obtained from natural henequen fibers. NFRCs and pristine PLA specimens were printed with a 0° raster angle for tension tests. The results showed that the NFRCs’ measured density, porosity, and degree of crystallinity increased with flour content. The tensile tests showed that the NFRC Young’s modulus was lower than that of the printed pristine PLA. For 1 wt% flour content, the NFRCs’ maximum stress and strain to failure were higher than those of the printed PLA, which was attributed to the henequen fibers acting as reinforcement and delaying crack growth. However, for 2 wt% and higher flour contents, the NFRCs’ maximum stress was lower than that of the printed PLA. Microscopic characterization after testing showed an increase in voids and defects, with the increase in flour content attributed to particle agglomeration. For 1 wt% flour content, the NFRCs were also printed with raster angles of ±45° and 90° for comparison; the highest tensile properties were obtained with a 0° raster angle. Finally, adding 3 wt% content of maleic anhydride to the NFRC with 1 wt% flour content slightly increased the maximum stress. The results presented herein warrant further research to fully understand the mechanical properties of printed NFRCs made of PLA reinforced with natural henequen fibers. Additive manufacturing (AM), also known as 3D printing, has been increasingly used in the last decade due to its versatility in producing numerous products with complex shapes and specific mechanical properties at a low cost. The increase in the use of 3D printing over the last few years has been driven by the rise in 3D printers’ affordability and the availability of printing materials . Three-dComposites from renewable resources based on FDM technology have become attractive products for construction , automobAgave fourcroydes Lem.) to reinforce 3D printed NFRC has not previously been reported to the authors’ knowledge. The henequen plant is endemic to the Yucatan peninsula in Mexico. Henequen fibers are lignocellulose fibers with a high cellulose content (60%) extracted from the leaves of the henequen plant. These fibers are used to fabricate ropes, mats, sacks, and bags [Based on the literature mentioned above, studies addressing the tensile properties of 3D printed PLA-based composites reinforced with natural fibers are scarce. Moreover, research on using henequen fibers , a melting temperature of 210 °C, a tensile elastic modulus of 3.5 GPa, and a tensile strength of 60 MPa [Extrusion-grade PLA Ingeo 2003D in pellet form  derived from renewable resources was used in this work. According to the manufacturer, the PLA material has a density of 1240 kg/mf 60 MPa .Long henequen fibers a were obBefore filament fabrication, PLA pellets were dried in an oven at 90 °C for 2 h, while the henequen flour was dried at 105 °C for 24 h. PLA/henequen NFRCs were prepared using a reinforcement content of 1–5 wt%. 2 in the gauge region. In addition, specimens with a PLA/H1 formulation  a at a temulation  were primulation c.Morphological observations were carried out by scanning electron microscopy (SEM) using an SEM microscope Jeol JSM 6360LV . All samples were coated with a thin layer of gold for electron conductivity. In addition, SEM observations were conducted on tested specimens to observe the fracture characteristics.P of the printed PLA and NFRCs was calculated using the following equation:3), 3 [Printed pristine PLA and NFRCs densities were measured using a Techne density gradient column  filled with an aqueous solution of calcium nitrate at 23 °C ,46,47. T70 kg/m3 , and WPLPLAW is the weight fraction of PLA in the sample.A differential scanning calorimetry (DSC) analysis of printed PLA and NFRCs was performed using a DSC 7 calorimeter  to study NFRCs’ melting behavior and crystallinity. Only the first heating was performed from room temperature to 220 °C at a heating rate of 5 °C/min under an inert nitrogen atmosphere. The cold crystallization temperature  history ,49. Xc wline PLA , and WPLUniaxial tensile tests were performed using a universal testing machine Shimadzu AGX-10 , equipped with a 1 kN load cell at a crosshead speed of 1 mm/min at room temperature. The tensile tests were carried out according to the ASTM D638 standard.P ≤ 0.05. For the DSC analysis, only one sample of each type of printed material was tested; thus, no statistical analysis of the DSC results is presented.The results are presented as the mean ± standard deviation of at least three repetitions. Statistical analysis was carried out by a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, which was performed using the stats.tukey_hsd function from the SciPy Python package . It was 3) than the PLA (1240 kg/m3). However, it can also be seen that the measured density is lower than the theoretical density, which is also expected considering the voids between adjacent layers and deposited beads generated during the printing process , there is an increase in porosity and void formation, which may be attributed to particle agglomeration resulting in a reduction in mechanical properties. However, further studies should be performed to confirm these observations and to understand how flour content affects the overall performance of the NRCFs. A careful selection of particle sizes and a double extrusion process  could inThe SEM images showed that the NFRCs exhibited debonding between adjacent deposited beads, mainly attributed to interfacial defects and voids formed during the printing process; these defects ultimately acted as stress concentrators and points of failure initiation. The presence of voids and bead shrinkage caused by the semicrystalline nature of the PLA  may haveIt has been reported that the infill pattern used during the 3D printing of PLA specimens affects their mechanical properties . For insTherefore, it is recommended to investigate different parameters that could improve the printing quality and influence the printed NFRCs’ mechanical performance, such as processing temperatures, layer orientation, deposition speed, infill pattern, and layer thickness . FurtherThe henequen fiber flour used to fabricate the NFRCs consisted of particles with sizes between 90–250 μm comprising a more significant percentage (67%) of small particles (90–150 μm).The measured density and porosity of the NFRCs increased with the increase in flour content.Thermal analysis by DSC showed that adding henequen flour to the PLA matrix increased the NFRCs’ degree of crystallinity.The Young’s modulus of the NFRCs was lower than that of the printed pristine PLA, which was attributed to particle agglomeration and voids.The maximum stress and strain to failure of the NFRC with 1% flour content were higher than those of the printed PLA, attributed to the henequen fibers acting as reinforcement and delaying crack growth.However, for 2% and higher flour contents, the NFRCs’ maximum stress was lower than those of the printed pristine PLA and PLA with 1% flour content.Microscopic characterization of NFRCs after tensile testing showed an increase in voids and defects with the increase in flour content, which was attributed to particle agglomeration in the material.For 1 wt% flour content, the highest tensile properties were obtained with a 0° raster angle.Adding 3 wt% content of maleic anhydride to the NFRC with 1 wt% flour content slightly increased the maximum stress.The mechanical results indicated that printed NFRCs made of PLA reinforced with henequen fiber have the potential to be used as sustainable materials with improved mechanical properties; however, further research is still required to understand the mechanical performance of these materials fully.In this work, natural fiber-reinforced composite (NFRC) filaments for 3D printing were fabricated using PLA reinforced with 1–5 wt% flour from henequen fibers. The filaments were subsequently used for the 3D printing of specimens for tension tests with a 0° raster angle. The effect of the flour content on the tensile properties was evaluated. Thermal, physical, and microscopic characterizations of the printed materials were also performed. In addition, for 1 wt% flour content, the NFRCs were also printed with raster angles of ±45° and 90°. Finally, an NFRC formulation with 3 wt% content of maleic anhydride and 1 wt% flour content was investigated. The following conclusions can be drawn from the results of this study: