Search Results (50)

For the large-scale fusion magnets of the International Thermonuclear Experimental Reactor (ITER) tokamak, wound with cable-in-conduit conductors, the application of sophisticated numerical models able to analyse the thermal–hydraulic behaviour during plasma scenarios is of paramount importance to guarantee an adequate stability margin during operating conditions. The SuperMagnet code has been developed by CryoSoft with the intent to simultaneously simulate the electrical, thermal and hydraulic phenomena occurring during the operation of superconducting coils. In this work, the SuperMagnet code is applied to analyse the thermal–hydraulic behaviour of the central solenoid of the ITER tokamak under the plasma scenario. The central solenoid (CS) is composed of six modules for a total amount of 240 pancakes. The software is able to tackle the complex structure of the CS and its cryogenic closed loop. In the present work, the circulation pump operation and the heat transfer to the helium bath are investigated. The results presented here show the temperature evolution of the magnet and of the supercritical helium during the plasma scenario, which allows the determination of the operation margin of the CS. Full article

Phosphoglucomutase (EC 5.4.2.2., PGM), a key enzyme of glycogenolysis and glycogenesis, catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate, whereas phosphomannomutase (EC 5.4.2.8., PMM) transfers the phosphate group from the 1′ to the 6′, or from the 6′ to the 1′ position in mannose phosphate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, a single gene, Tk1108, encodes a protein with both PGM and PMM activities. Here, we report biophysical analysis and the 2.45 Å resolution cryo-EM structure of this novel enzyme. Our results demonstrate a specific arrangement of the four subunits in the quaternary structure, displaying a distinct catalytic cleft required for the bifunctional activity at extremely high temperatures. To the best of our knowledge, this is the first biophysical characterization and cryo-EM structure elucidation of a thermostable, bifunctional PGM/PMM. Full article

Ctr1 is a membrane-spanning homotrimer that facilitates copper uptake in eukaryotic cells with high affinity. While structural details of the transmembrane domain of human Ctr1 have been elucidated using X-ray crystallography and cryo-EM, the transfer mechanisms of copper and the conformational changes that control the gating mechanism remain poorly understood. The role of the extracellular N-terminal domains is particularly unclear due to the absence of a high-resolution structure of the full-length hCtr1 protein and limited biochemical and biophysical characterization of the transporter in solution and in cell. In this study, we employed distance electron paramagnetic resonance to investigate the conformational changes of the extracellular N-terminal domain of full-length hCtr1, both in vitro and in cells, as a function of Cu(I) binding. Our results demonstrate that at specific Cu(I) concentrations, the extracellular chains move closer to the lumen to facilitate copper transfer. Additionally, while at these concentrations the intracellular part is penetrating the lumen, suggesting a ball-and-chain gating mechanism. Moreover, this phenomenon was observed for both reconstituted protein in micelles and in native cell membranes. However, the measured distance values were slightly different, suggesting that the membrane’s characteristics and therefore its lipid composition also impact and even regulate the gating mechanism of hCtr1. Full article

Background/Objectives: The endosomal escape of lipid nanoparticles (LNPs) is crucial for efficient mRNA-based therapeutics. Here, we present a cationic polymeric micelle (cPM) as a safe and potent co-delivery system with enhanced endosomal escape capabilities. Methods: We synthesized a cationic and ampholytic di-block copolymer, poly (poly (ethylene glycol)_4-5 methacrylate_a-co-hexyl methacrylate_b)_X-b-poly(butyl methacrylate_c-co-dimethylaminoethyl methacrylate_d-co-propyl acrylate_e)_Y (p(PEG_4-5MA_a-co-HMA_b)_X-b-p(BMA_c-co-DMAEMA_d-co-PAA_e)_Y), via reversible addition–fragmentation chain transfer polymerization. The cPMs were then formulated using the synthesized polymer by the dispersion–diffusion method and characterized by dynamic light scattering (DLS) and cryo-transmission electron microscopy (CryoTEM). The membrane-destabilization activity of the cPMs was evaluated by a hemolysis assay. We performed an in vivo functional assay of firefly luciferase (Fluc) mRNA using two of the most commonly studied LNPs, SM102 LNP and Dlin-MC3-DMA LNPs. Results: With a particle size of 61.31 ± 0.68 nm and a zeta potential of 37.76 ± 2.18 mV, the cPMs exhibited a 2–3 times higher firefly luciferase signal at the injection site compared to the control groups without cPMs following intramuscular injection in mice, indicating the high potential of cPMs to enhance the endosomal escape efficiency of mRNA-LNPs. Conclusions: The developed cPM, with enhanced endosomal escape capabilities, presents a promising strategy to improve the expression efficiency of delivered mRNAs. This approach offers a novel alternative strategy with no modifications to the inherent properties of mRNA-LNPs, preventing any unforeseeable changes in formulation characteristics. Consequently, this polymer-based nanomaterial holds immense potential for clinical applications in mRNA-based vaccines. Full article

This paper presents Cryo-SIMPLY, a reliable smart material implication (SIMPLY) operating at cryogenic conditions (77 K). The assessment considers SIMPLY schemes based on spin-transfer torque magnetic random access memory (STT-MRAM) technology with single-barrier magnetic tunnel junction (SMTJ) and double-barrier magnetic tunnel junction (DMTJ). Our study relies on a temperature-aware macrospin-based Verilog-A compact model for MTJ devices and a 65 nm commercial process design kit (PDK) calibrated down to 77 K under silicon measurements. The DMTJ-based SIMPLY demonstrates a significant improvement in read margin at 77 K, overcoming the conventional SIMPLY scheme at room temperature (300 K) by approximately 2.3 X. When implementing logic operations with the SIMPLY scheme operating at 77 K, the DMTJ-based scheme assures energy savings of about 69%, as compared to its SMTJ-based counterpart operating at 77 K. Overall, our results prove that the SIMPLY scheme at cryogenic conditions is a promising solution for reliable and energy-efficient logic-in-memory (LIM) architectures. Full article

Proton-translocating NADH–ubiquinone oxidoreductase (complex I) catalyzes the oxidation of NADH by ubiquinone accompanied by the transmembrane transfer of four protons, thus contributing to the formation of a proton motive force (pmf) across the coupling membranes of mitochondria and bacteria, which drives ATP synthesis in oxidative phosphorylation. In recent years, great progress has been achieved in resolving complex I structure by means of X-ray crystallography and high-resolution cryo-electron microscopy, which has led to the formulation of detailed hypotheses concerning the molecular mechanism of coupling of the redox reaction to vectorial proton translocation. To test and probe proposed mechanisms, a comprehensive study of complex I using other methods including molecular dynamics and a variety of biochemical studies such as kinetic and inhibitory analysis is required. Due to complex I being a major electron entry point for oxidative metabolism, various mutations of the enzyme lead to the development of severe pathologies and/or are associated with human metabolic disorders and have been well documented. This review examines current information on the structure and subunit composition of complex I of eukaryotes and prokaryotes, reactions catalyzed by this enzyme, and ways to regulate them. The review also discusses biomedical aspects related to the enzyme in light of recent findings. Full article

Plastocyanin is a small mobile protein that facilitates electron transfer through the formation of short-lived protein–protein complexes with cytochrome bf and photosystem 1. Due to the transient nature of plastocyanin–cytochrome f complex, the lack of a long-lived tight complex makes it impossible to determine its structure by X-ray diffraction analysis. Up to today, a number of slightly different structures of such complexes have been obtained by experimental and computer methods. Now, artificial intelligence gives us the possibility to predict the structures of intermolecular complexes. In this study, we compare encounter and final complexes obtained by Brownian and molecular dynamics methods, as well as the structures predicted by AlphaFold 3, with NMR and cryo-EM data. Surprisingly, the best match for the plastocyanin electron density obtained by cryo-EM was demonstrated by an AlphaFold 3 structure. The orientation of plastocyanin in this structure almost completely coincides with its orientation obtained by molecular dynamics calculation, and, at the same time, it is different from the orientation of plastocyanin predicted on the basis of NMR data. This is even more unexpected given that only NMR structures for the plastocyanin-cytochrome f complex are available in the PDB database, which was used to train AlphaFold 3. Full article

A Type I reaction center (RC) (Fe-S type, ferredoxin reducing) is found in several phyla containing anoxygenic phototrophic bacteria. These include the heliobacteria (HB), the green sulfur bacteria (GSB), and the chloracidobacteria (CB), for which high-resolution homodimeric RC-photosystem (PS) structures have recently appeared. The 2.2-Å X-ray structure of the RC-PS of Heliomicrobium modesticaldum revealed that the core PshA apoprotein (PshA-1 and PshA-2 homodimeric pair) exhibits a structurally conserved PSI arrangement comprising five C-terminal transmembrane α-helices (TMHs) forming the RC domain and six N-terminal TMHs coordinating the light-harvesting (LH) pigments. The Hmi. modesticaldum structure lacked quinone molecules, indicating that electrons were transferred directly from the A₀ (8¹-OH-chlorophyll (Chl) a) acceptor to the F_X [4Fe-4S] component, serving as the terminal RC acceptor. A pair of additional TMHs designated as Psh X were also found that function as a low-energy antenna. The 2.5-Å resolution cryo-electron microscopy (cryo-EM) structure for the RC-PS of the green sulfur bacterium Chlorobaculum tepidum included a pair of Fenna–Matthews–Olson protein (FMO) antennae, which transfer excitations from the chlorosomes to the RC-PS (PscA-1 and PscA-2) core. A pair of cytochromes c_Z (PscC) molecules was also revealed, acting as electron donors to the RC bacteriochlorophyll (BChl) a’ special pair, as well as PscB, housing the [4Fe-4S] cluster F_A and F_B, and the associated PscD protein. While the FMO components were missing from the 2.6-Å cryo-EM structure of the Zn- (BChl) a’ special pair containing RC-PS of Chloracidobacterium thermophilum, a unique architecture was revealed that besides the (PscA)₂ core, consisted of seven additional subunits including PscZ in place of PscD, the PscX and PscY cytochrome c serial electron donors and four low mol. wt. subunits of unknown function. Overall, these diverse structures have revealed that (i) the HB RC-PS is the simplest light–energy transducing complex yet isolated and represents the closest known homolog to a common homodimeric RC-PS ancestor; (ii) the symmetrically localized Ca²⁺-binding sites found in each of the Type I homodimeric RC-PS structures likely gave rise to the analogously positioned Mn₄CaO₅ cluster of the PSII RC and the Tyr_Z RC donor site; (iii) a close relationship between the GSB RC-PS and the PSII Chl proteins (CP)43 and CP47 was demonstrated by their strongly conserved LH-(B)Chl localizations; (iv) LH-BChls of the GSB-RC-PS are also localized in the conserved RC-associated positions of the PSII Chl_Z-D1 and Chl_Z-D2 sites; (v) glycosylated carotenoids of the GSB RC-PS are located in the homologous carotenoid-containing positions of PSII, reflecting an O₂-tolerance mechanism capable of sustaining early stages in the evolution of oxygenic photosynthesis. In addition to the close relationships found between the homodimeric RC-PS and PSII, duplication of the gene encoding the ancestral Type I RC apoprotein, followed by genetic divergence, may well account for the appearance of the heterodimeric Type I and Type II RCs of the extant oxygenic phototrophs. Accordingly, the long-held view that PSII arose from the anoxygenic Type II RC is now found to be contrary to the new evidence provided by Type I RC-PS homodimer structures, indicating that the evolutionary origins of anoxygenic Type II RCs, along with their distinct antenna rings are likely to have been preceded by the events that gave rise to their oxygenic counterparts. Full article

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A “linear” 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect. Full article

Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters. Emerging themes include the following: (i) The obligatory coupling of protein and electron transfers does not occur in synthetic and small-molecule iron–sulfur clusters. The coupling of these processes in nitrogenase suggests that they may involve unique features of the cofactor, such as hydride formation on the trigonal prismatic arrangement of irons, protonation of belt sulfurs, and/or protonation of the interstitial carbon. (ii) Both the active site cofactor and protein are dynamic under turnover conditions; the changes are such that more highly reduced forms may differ in key ways from the resting-state structure. Homocitrate appears to play a key role in coupling cofactor and protein dynamics. (iii) Structural asymmetries are observed in nitrogenase under turnover-relevant conditions by cryo-electron microscopy, although the mechanistic relevance of these states (such as half-of-sites reactivity) remains to be established. Full article

Vegetatively propagating aquatic angiosperms, the Lemnaceae family (duckweeds) represents valuable genetic resources for circular bioeconomics and other sustainable applications. Due to extremely fast growth and laborious cultivation of in vitro collections, duckweeds are an urgent subject for cryopreservation. We developed a robust and fast DMSO-free protocol for duckweed cryopreservation by vitrification. A single-use device was designed for sampling of duckweed fronds from donor culture, further spin-drying, and subsequent transferring to cryo-tubes with plant vitrification solution 3 (PVS3). Following cultivation in darkness and applying elevated temperatures during early regrowth stage, a specific pulsed illumination instead of a diurnal regime enabled successful regrowth after the cryopreservation of 21 accessions of Spirodela, Landoltia, Lemna, and Wolffia genera, including interspecific hybrids, auto- and allopolyploids. Genome size measurements revealed no quantitative genomic changes potentially caused by cryopreservation. The expression of CBF/DREB1 genes, considered as key factors in the development of freezing tolerance, was studied prior to cooling but was not linked with duckweed regrowth after rewarming. Despite preserving chlorophyll fluorescence after rewarming, the rewarmed fronds demonstrated nearly zero photosynthetic activity, which did not recover. The novel protocol provides the basis for future routine application of cryostorage to duckweed germplasm collections, saving labor for in vitro cultivation and maintaining characterized reference and mutant samples. Full article

Along with the adoption of three-dimensional electron diffraction (3D ED/MicroED) as a mainstream tool for structure determination from sub-micron single crystals, questions about best practices regarding each step along the workflow, from data collection to structure solutions, arise. In this paper, we discuss three particular aspects of a 3D ED/MicroED experiment which, after hundreds of structures solved in Rigaku’s laboratories, we have found to be important to consider carefully. First, for a representative model system of a hydrated compound (trehalose dihydrate), we show that cryo-transfer of the sample into the diffractometer is an effective means to prevent dehydration, while cooling of the sample without cryo-transfer yields a marginal improvement only. Next, we demonstrate for a small (tyrosine) and a large (clarithromycin) organic compound, how a simplified and fast workflow for dynamical diffraction calculations can determine absolute crystal structures with high confidence. Finally, we discuss considerations and trade-offs for choosing an optimal effective crystal-to-detector distance; while a long distance is mandatory for a protein (thaumatin) example, even a small molecule with difficult diffraction behavior (cystine) yields superior results at longer distances than the one used by default. Full article

Green alga Chlorella ohadii is known for its ability to carry out photosynthesis under harsh conditions. Using cryogenic electron microscopy (cryoEM), we obtained a high-resolution structure of PSII at 2.72 Å. This structure revealed 64 subunits, which encompassed 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, a unique subunit arrangement was observed to protect the oxygen-evolving complex. This arrangement involved PsbO (OEE1), PsbP (OEE2), PsbB, and PsbU (a homolog of plant OEE3). PsbU interacted with PsbO, PsbC, and PsbP, thereby stabilizing the shield of the oxygen-evolving complex. Significant changes were also observed at the stromal electron acceptor side. PsbY, identified as a transmembrane helix, was situated alongside PsbF and PsbE, which enclosed cytochrome b559. Supported by the adjacent C-terminal helix of Psb10, these four transmembrane helices formed a bundle that shielded cytochrome b559 from the surrounding solvent. Moreover, the bulk of Psb10 formed a protective cap, which safeguarded the quinone site and likely contributed to the stacking of PSII complexes. Based on our findings, we propose a protective mechanism that prevents Q_B (plastoquinone B) from becoming fully reduced. This mechanism offers insights into the regulation of electron transfer within PSII. Full article

This article presents data from experiments to determine the cryoresistance of Charollais sheep embryos, depending on the stage of embryo development and the method of freezing, as well as the results of embryo transfer. The study design consisted of a study on the cryopreservation of ewe embryos at different developmental stages (early, 2–8 blastomeric and late, at the morula/blastocyst stage), two cryopreservation protocols (slow freezing and ultra-fast vitrification), and embryo transfer of cryo- and fresh embryos. Embryos from Charollais sheep donors (n = 12) were recovered after induction of superovulation. The embryos were recovered surgically (laparotomy) on days 2 and 6 after insemination. Before there was transfer to recipients, part of embryos was cryopreserved using standard slow freezing and ultra-fast vitrification methods. The average ovarian response was 7.54 ovulations per donor, and 5.83 embryos per donor were collected. No effect of the cryopreservation method and embryo development stage on the preservation of the morphological structure of embryos was found. There were no significant differences in the survival rate of cryoembryos at different development stages, frozen using different techniques, and after transfer to recipients. Differences in cryoresistance between embryonic developmental stages in favor of the morula/blastocyst stage were found (survival after thawing 86.4% vs. 75.0% in early embryos). At different stages of development, the survival rate of fresh embryos (45.8%) compared to cryopreserved ones (30.2%) was significantly higher (p < 0.05), while among fresh ones, the best survival rate (50.0%) was observed after the transfer of morules and blastocysts. Full article

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations. Full article