Structural insights into vesicular monoamine storage and drug interactions.

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Identifying Heterozipper ß-Sheet in Twisted Amyloid Aggregation.

Amyloid peptide (AP) self-assembly is a hierarchical process. However, the mechanistic rule of guiding peptides to organize well-ordered nanostructure in a clear and precise manner remains poorly understood. Herein we explored the molecular insight of AP motif aggregates underlying hierarchical process with helical fibrillar structure by atomic force microscope, cryo-electron microscopy (cryo-EM), and molecular dynamics simulation. AP assembly encompasses well-ordered twisted fibrils with uniform morphology, size, and periodicity. More importantly, a heterozipper ß-sheet was identified in a protofilament of AP assembly determined by cryo-EM with a high resolution of 3.5 Å. Each peptide heterozipper was further composed of two antiparallel ß strands and arranged by an alternative manner in a protofilament. The hydrophobic core and hydrophilic area in each zipper played the significant role for peptide assembling. This work proposed and verified the rule facilitating the basic building unit to form twisted fibrils and gave the explanation of peptide hierarchical assembling.

MADS2 regulates priming defence in postharvest peach through combined salicylic acid and abscisic acid signaling.

MADS-box genes play well-documented roles in plant development, but relatively little is known regarding their involvement in defence responses. In this study, pre-treatment of peach (Prunus persica) fruit with ß-aminobutyric acid (BABA) activated resistance against Rhizopus stolonifer, leading to a significant delay in the symptomatic appearance of disease. This was associated with an integrated defence response that included a H2O2 burst, ABA accumulation, and callose deposition. cDNA library screening identified nucleus-localized MADS2 as an interacting partner with NPR1, and this was further confirmed by yeast two-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays. The DNA-binding activity of NPR1 conferred by the NPR1-MADS2 complex was required for the transcription of SA-dependent pathogenesis-related (PR) and ABA-inducible CalS genes in order to gain the BABA-induced resistance, in which MAPK1-induced post-translational modification of MADS2 was also involved. In accordance with this, overexpression of PpMADS2 in Arabidopsis potentiated the transcription of a group of PR genes and conferred fungal resistance in the transgenic plants. Conversely, Arabidopsis mads2-knockout lines showed high sensitivity to the fungal pathogen. Our results indicate that MADS2 positively participates in BABA-elicited defence in peach through a combination of SA-dependent NPR1 activation and ABA signaling-induced callose accumulation, and that this defence is also related to the post-translational modification of MADS2 by MAPK1 for signal amplification.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.

Composition and processing activity of a semi-recombinant holo U7 snRNP.

In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3' end processing of histone pre-mRNAs.

Alterations in Sucrose and Phenylpropanoid Metabolism Affected by BABA-Primed Defense in Postharvest Grapes and the Associated Transcriptional Mechanism.

Defense elicitors can induce fruit disease resistance to control postharvest decay but may incur quality impairment. Our present work aimed to investigate the resistance against Botrytis cinerea induced by the elicitor ß-aminobutyric acid (BABA) and to elucidate the specific transcriptional mechanism implicated in defense-related metabolic regulations. The functional dissection results demonstrated that, after inoculation with the fungal necrotroph B. cinerea, a suite of critical genes encoding enzymes related to the sucrose metabolism and phenylpropanoid pathway in priming defense in grapes were transcriptionally induced by treatment with 10 mM BABA. In contrast, more UDP-glucose, a shared precursor of phenylpropanoid and sucrose metabolism, may be redirected to the phenylpropanoid pathway for the synthesis of phytoalexins, including trans-resveratrol and ɛ-viniferin, in 100 mM BABA-treated grapes, resulting in direct resistance but compromised soluble sugar contents. An R2R3-type MYB protein from Vitis vinifera, VvMYB44, was isolated and characterized. VvMYB44 expression was significantly induced upon the grapes expressed defensive reaction. Subcellular localization, yeast two-hybrid, and coimmunoprecipitation assays revealed that the nuclear-localized VvMYB44 physically interacted with the salicylic acid-responsive transcription coactivator NPR1 in vivo for defense expression. In addition, VvMYB44 directly bound to the promoter regions of sucrose and phenylpropanoid metabolism-related genes and transactivated their expression, thus tipping the balance of antifungal compound accumulation and soluble sugar maintenance. Hence, these results suggest that 2R-type VvMYB44 might be a potential positive participant in BABA-induced priming defense in grape berries that contributes to avoiding the excessive consumption of soluble sugars during the postharvest storage.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dual function of VvWRKY18 transcription factor in the ß-aminobutyric acid-activated priming defense in grapes.

Induction of phytoalexin production after invading pathogens is recognized as an essential aspect of the plant-induced resistance. The WRKY family includes plant-specific transcriptional factors associated with plant defense responses, but the comprehensive mechanisms are poorly understood. Here, we attempted to elaborate the regulatory function of VvWRKY18 from the group IIa of WRKY transcription factor (TF) from Vitis vinifera, in the regulation of ß-aminobutyric acid (BABA)-activated stilbene phytoalexins biosynthesis and PATHOGENESIS-RELATED (PR) genes expressions in grapes. BABA at 10 mmol L-1 triggered a priming protection in grapes and conferred a potentiation of the expression levels of VvWRKY18, VvNPR1, and several salicylic acid (SA)-responsive genes, which was accompanied by enhanced stilbene production upon Botrytis cinerea infection. In addition, a physical interaction between VvWRKY18 and the regulatory protein VvNPR1 was detected in vivo and in vitro by yeast-2-hybrid (Y2H), pull-down and co-immunoprecipitation assay (Co-IP) assays. Furthermore, yeast-1-hybrid (Y1H) and dual-luciferase reporter (DLR) assays indicated that VvWRKY18 activated the transcription of STILBENE SYNTHASE (STS) genes, including VvSTS1 and VvSTS2, by directly binding the W-box elements within the specific promoters and resultantly enhancing stilbene phytoalexins biosynthesis. Further investigation demonstrated that heterologous expression of VvWRKY18 elevated the transcriptions of STS and PR genes, thus contributing to potentiating the defense of transgenic Arabidopsis thaliana plants and resultantly inhibiting B. cinerea invasion. Hence, VvWRKY18 serves as a singular effector involved in the synthesis of stilbene phytoalexins in grapes and its interaction with VvNPR1 provided DNA binding ability required for VvNPR1 to initiate systemic acquired resistance (SAR) defense.

Redox status regulates subcelluar localization of PpTGA1 associated with a BABA-induced priming defence against Rhizopus rot in peach fruit.

This study attempted to characterize the involvement of a change in the redox status and subcellular localization in the BABA-induced priming resistance of peach fruit against Rhizopus rot. Specifically, 50 mM BABA primed the peaches for the enhanced disease resistance against R. stolonifer, as demonstrated by suppression of the disease development upon pathogen challenge accompanied by the clearly elevated level of TGA transcription factor (PpTGA1) and NPR1 gene (PpNPR1). In addition, the BABA elicitation enhanced the activities of a series of critical enzymes in the PPP and AsA-GSH cycle, and eventually promoted the NADPH and GSH pools, which altered the intracellular redox state towards a highly reductive condition. Additionally, PpTGA1-GFP was localized in the cytoplasm in the absence of BABA treatment or R. stolonifer inoculation, while BABA elicitation plus R. stolonifer inoculation caused PpTGA1-GFP to specifically translocate to the nucleus, where it interacted with PpNPR1 and regulated the positive expression of PR genes. Therefore, the observations implied that BABA could promote the reduction of the redox state, resulting in the translocation of PpTGA1 to the nucleus, which was a prerequisite for the induction of a priming defence against Rhizopus rot in peach.

Structure and mechanism of Zn2+-transporting P-type ATPases.

Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn(2+)-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn(2+) and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·Pi) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively. The structures reveal a similar fold to Cu(+)-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·Pi state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn(2+) release as a built-in counter ion, as has been proposed for H(+)-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between PIB-type Zn(2+)-ATPases and PIII-type H(+)-ATPases and at the same time show structural features of the extracellular release pathway that resemble PII-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+), K(+)-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.

Potential modes of action of Pseudomonas fluorescens ZX during biocontrol of blue mold decay on postharvest citrus.

BACKGROUND: The application of chemical fungicides is currently the main method for the control of postharvest decay of fruits and vegetables. However, public concern has been growing towards the effect of fungicides on food safety, human health and environmental pollution. Thus, interest in microbial biocontrol agent development has grown, such agents being considered both safe and environmentally friendly. Pseudomonas fluorescens is widely distributed in nature, and one of the most valuable biocontrol and plant growth-promoting rhizobacteria. In this study, the efficacy and the potential associated modes of action of P. fluorescens ZX against Penicillium italicum on oranges (Citrus sinensis Osbeck) were investigated. RESULTS: The application of P. fluorescens ZX significantly reduced blue mold lesion size and incidence in comparison to the control, where P. fluorescens ZX was effective when applied preventatively but not curatively. In dual cultures, treatment with cell-free autoclaved cultures or culture filtrate had a limited capacity to suppress P. italicum, while P. italicum was inhibited by bacterial fluid and bacterial suspension with living cells in vitro. The P. fluorescens ZX isolate displayed protease, but not chitinase, glucanase or cellulose, activity, and produced siderophores and volatile organic compounds with antifungal abilities. Competition tests showed P. fluorescens ZX could use fructose, sucrose, aspartic acid, threonine, serine, glycine, valine, lysine and proline better than P. italicum. Furthermore, an effective biofilm that peaked after a 24-hour incubation at 30 °C was formed by the P. fluorescens ZX isolate. Light microscopy and scanning electron microscopy observations indicate the P. fluorescens ZX isolate could not undergo direct parasitism or hyperparasitism. CONCLUSIONS: Competition for nutrients and niches, biofilm formation, inhibition of spore germination and mycelial growth, and production of inhibitory metabolites may play important roles in P. fluorescens ZX antagonism of P. italicum. © 2019 Society of Chemical Industry.

Structure and Function of Cu(I)- and Zn(II)-ATPases.

Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing the molecular differences that allow transport of these two distinct metal types, and discuss possible future directions of research in the field.

Structure of the bifunctional methyltransferase YcbY (RlmKL) that adds the m7G2069 and m2G2445 modifications in Escherichia coli 23S rRNA.

The 23S rRNA nucleotide m(2)G2445 is highly conserved in bacteria, and in Escherichia coli this modification is added by the enzyme YcbY. With lengths of around 700 amino acids, YcbY orthologs are the largest rRNA methyltransferases identified in Gram-negative bacteria, and they appear to be fusions from two separate proteins found in Gram-positives. The crystal structures described here show that both the N- and C-terminal halves of E. coli YcbY have a methyltransferase active site and their folding patterns respectively resemble the Streptococcus mutans proteins Smu472 and Smu776. Mass spectrometric analyses of 23S rRNAs showed that the N-terminal region of YcbY and Smu472 are functionally equivalent and add the m(2)G2445 modification, while the C-terminal region of YcbY is responsible for the m(7)G2069 methylation on the opposite side of the same helix (H74). Smu776 does not target G2069, and this nucleotide remains unmodified in Gram-positive rRNAs. The E.coli YcbY enzyme is the first example of a methyltransferase catalyzing two mechanistically different types of RNA modification, and has been renamed as the Ribosomal large subunit methyltransferase, RlmKL. Our structural and functional data provide insights into how this bifunctional enzyme evolved.

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases.

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD-1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD-1, MBD-2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Maintaining quality and bioactive compounds of broccoli by combined treatment with 1-methylcyclopropene and 6-benzylaminopurine.

BACKGROUND: Broccoli deteriorates very quickly after harvest at ambient temperature due to the loss of green colour and the consequent yellowing of florets. To search for an effective method to control quality deterioration, the effect of 1-methylcyclopropene (1-MCP) combined with 6-benzylaminopurine (6-BA) treatment on visual quality, antioxidant enzymes and bioactive compounds in broccoli florets were investigated. RESULTS: A combined treatment of 2.5 µL L⁻¹ 1-MCP and 200 mg L⁻¹ 6-BA significantly reduced the increase of lightness (L*) value, and retained a high level for the hue value (H) and chlorophyll content. Superoxide dismutase, ascobate peroxidase and catalase activities increased while the activity of peroxidase decreased during storage in treated samples in comparison with the controls. The combined treatment enhanced the biosynthesis of glucosinolate and the formation of the anticarcinogen sulforaphane, which improved the health benefit of broccoli. CONCLUSION: These results indicate that a combined treatment of 1-MCP and 6-BA could be a good candidate for maintaining the visual quality and enhancing the nutritional value in broccoli during storage at 15 °C.

Cryo-EM structure of the trehalose monomycolate transporter, MmpL3, reconstituted into peptidiscs.

Mycobacteria have an atypical thick and waxy cell wall. One of the major building blocks of such mycomembrane is trehalose monomycolate (TMM). TMM is a mycolic acid ester of trehalose that possesses long acyl chains with up to 90 carbon atoms. TMM represents an essential component of mycobacteria and is synthesized in the cytoplasm, and then flipped over the plasma membrane by a specific transporter known as MmpL3. Over the last decade, MmpL3 has emerged as an attractive drug target to combat mycobacterial infections. Recent three-dimensional structures of MmpL3 determined by X-ray crystallography and cryo-EM have increased our understanding of the TMM transport, and the mode of action of inhibiting compounds. These structures were obtained in the presence of detergent and/or in a lipidic environment. In this study, we demonstrate the possibility of obtaining a high-quality cryo-EM structure of MmpL3 without any presence of detergent through the reconstitution of the protein into peptidiscs. The structure was determined at an overall resolution of 3.2 Å and demonstrates that the overall structure of MmpL3 is preserved as compared to previous structures. Further, the study identified a new structural arrangement of the linker that fuses the two subdomains of the transmembrane domain, suggesting the feature may serve a role in the transport process.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy - rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

PpMYB1 and PpNPR1 interact to enhance the resistance of peach fruit to Rhizopus stolonifer infection.

MYB transcription factors play important role in stress-resistance of plants. Nevertheless, the function of MYB TFs in peach Rhizopus rot remains poorly understood. Herein, Pichia guilliermondii treatment activated resistance against Rhizopus stolonifer, as illustrated by reductions in the incidence rate and severity of Rhizopus rot disease, increased enzyme activities and gene expression of chitinase (CHI) and ß-1,3-glucanase (GLU), and enhancement of energy production by inducing the activities and expression of H+-ATPase and Ca2+-ATPase, succinate dehydrogenase (SDH), and cytochrome c oxidase (CCO). Moreover, an R1-type MYB, PpMYB1, from peach fruit was induced during R. stolonifer infection and in response to P. guilliermondii treatment. PpMYB1 activated the transcription of PpCHI-EP3 and PpGLU-like genes and the energy metabolism-related gene PpH+-ATPase1 by directly targeting the MBS element. Importantly, PpMYB1 interacted with PpNPR1 to form a heterodimer, which was conducive to enhancing the activation of target gene transcription. Collectively, our findings suggest that PpMYB1 cooperates with PpNPR1 to positively regulate disease resistance by activating the disease defense system and energy metabolism in peaches.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM.

Different nanobubbles mitigate cadmium toxicity and accumulation of rice (Oryza sativa L.) seedlings in hydroponic cultures.

Cadmium (Cd) contamination can pose a severe threat to food production and human health. The accumulation of Cd in rice will decrease rice biomass, photosynthetic activity, and antioxidant capacity, affecting crop yield. The effects of different nanobubbles on the growth and Cd accumulation of rice seedlings under hydroponic conditions were investigated in this study. The results showed that the biomass, photosynthetic pigment content, and antioxidant enzyme activity of rice seedlings decreased when treated with Cd alone and that Cd induced lipid peroxidation in rice seedlings. However, when different types of nanobubbles were introduced into the nutrient solution, the bioavailability of Cd in the solution was reduced. As a result, the Cd content in rice was significantly decreased compared to treatment with Cd alone. Nanobubbles increased the biomass of rice, enhanced photosynthesis, and improved the antioxidant capacity of rice by increasing antioxidant enzyme activities to alleviate Cd-induced oxidative stress. At the same time, nanobubbles increased the Fe content in rice, which decreased the Cd content, as Cd is antagonistic to Fe. In conclusion, these results suggested that nanobubbles are a potential method of mitigating Cd stress that may help to improve rice yield and could be further explored in production.