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Bacterial microcompartment assembly: The key role of encapsulation peptides

Bacterial microcompartments (BMCs) are proteinaceous organelles used by a broad range of bacteria to segregate and optimize metabolic reactions. Their functions are diverse, and can be divided into anabolic (carboxysome) and catabolic (metabolosomes) processes, depending on their cargo enzymes. The assembly pathway for the β-carboxysome has been characterized, revealing that biogenesis proceeds from the inside out. The enzymes coalesce into a procarboxysome, followed by encapsulation in a protein shell that is recruited to the procarboxysome by a short (~17 amino acids) extension on the C-terminus of one of the encapsulated proteins. A similar extension is also found on the N- or C-termini of a subset of metabolosome core enzymes. These encapsulation peptides (EPs) are characterized by a primary structure predicted to form an amphipathic α-helix that interacts with shell proteins. In this study, we review the features, function and widespread occurrence of EPs among metabolosomes, and propose an expanded role for EPs in the assembly of diverse BMCs.

59 BASIC BIOLOGICAL SCIENCES↗

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here in this work, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. In conclusion, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.

14-3-3↗

Neutron Scattering Studies of the Interplay of Amyloid β Peptide(1–40) and An Anionic Lipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction between lipid bilayers and Amyloid β peptide (Aβ) plays a critical role in proliferation of Alzheimer’s disease (AD). AD is expected to affect one in every 85 humans by 2050, and therefore, deciphering the interplay of Aβ and lipid bilayers at the molecular level is of profound importance. In this work, we applied an array of neutron scattering methods to study the structure and dynamics of Aβ(1–40) interacting 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) bilayers. In the structural investigations of lipid bilayer’s response to Aβ binding, Small Angle Neutron Scattering and Neutron Membrane Diffraction revealed that the Aβ anchors firmly to the highly charged DMPG bilayers in the interfacial region between water and hydrocarbon chain, and it doesn’t penetrate deeply into the bilayer. This association mode is substantiated by the dynamics studies with high resolution Quasi-Elastic Neutron Scattering experiments, showing that the addition of Aβ mainly affects the slower lateral motion of lipid molecules, especially in the fluid phase, but not the faster internal motion. The results revealed that Aβ associates with the highly charged membrane in surface with limited impact on the structure, but the altered membrane dynamics could have more influence on other membrane processes.

59 BASIC BIOLOGICAL SCIENCES↗

EMSL Community Science Campaign Meeting: Critical Minerals and Materials - Rhizo Critical Campaign Breakout Session Report Summary

The “Critical Minerals Biogeochemistry in the Rhizosphere – Ultramafic Soils (Rhizo Critical)” campaign breakout (BO) session was organized to identify major knowledge gaps and fundamental research needs in rhizosphere microbiology and geochemistry that, if addressed, could transform our ability to recover critical minerals from ultramafic soil systems. We sought to identify significant challenges that must be surmounted in the pursuit of deeper science knowledge. Our ultimate goal is to understand this landscape well enough to identify and prioritize opportunities for EMSL to make the greatest impact with Environmental Transformations and Interactions (ETI) science area research campaigns focused on the biogeochemical processes controlling the behavior of critical minerals and materials in the rhizosphere. The increasing demand for critical materials and minerals (CMM) in the U.S. has heightened interest in low-grade ores with much attention on ultramafic soils, which contain valuable metals such as nickel (Ni), chromium (Cr), manganese, cobalt (Co), and copper (Lee et al., 2025; DOE CMM Report, 2023) used in advanced battery, magnet, wiring and wind turbines, and stainless steel technologies. Metal hyperaccumulating plants grown in ultramafic soils can extract economically valuable concentrations of CMMs through the process of phytomining. This technology has evolved from phytoremediation, which involves using plants to cleanse contaminated environments by removing, detoxifying, or stabilizing pollutants like metals and organic compounds. Hyperaccumulator plants are capable of storing metals in their living tissues at concentrations hundreds to thousands of times higher than those found in 'normal' plants. For instance, while the average concentration of Ni in the dry matter of plants growing in typical soils is usually less than 5 µg g?¹, Ni hyperaccumulation is defined by concentrations exceeding 1,000 µg g?¹ (Corzo Remigio et al., 2020; Reeves et al., 2018). Phytomining research has primarily focused on Ni (Rylott and van der Ent, 2025), for which the U.S. has very limited conventional mines in operation. Most soils typically contain Ni concentrations ranging from 7 to 50 mg kg-1, whereas serpentine soils exhibit significantly higher levels, with Ni content often ranging between 700 and 8,000 mg kg-1 (Sobczyk et al., 2017). While more than 500 plant species in over 50 different families have been identified as Ni hyperaccumulators (Kidd et al., 2018), Ni phytomining (and phytominng in general) remains largely untested because most studies are short-term, small-scale, and conducted under simplified or artificially enriched conditions, so they fail to capture the low metal concentrations, environmental variability, and management constraints that would be needed for a field-scale demonstration. Few hyperaccumulator species have been validated as true “metal crops,” and their biomass production, stress tolerance, and rooting characteristics are usually too poor to yield economically meaningful metal outputs. Critically, the basic mechanisms of metal uptake, transport, and sequestration, especially as shaped by belowground processes such as root exudation, rhizosphere chemistry, and root–microbe interactions that control metal mobility and bioavailability (Montreemuk et al., 2023; Kidd et al., 2018; Durand et al., 2023; Alford et al., 2010), are still only partially understood, and downstream metal recovery from biomass is rarely optimized. Because these limitations stem from gaps in fundamental knowledge rather than from a failure of the concept itself (Rylott and van der Ent, 2025; van der Ent et al., 2015), there is a strong need for basic science that dissects plant metal homeostasis, rhizosphere and microbial processes, and their integration with soil chemistry and process engineering to design more robust, scalable phytomining systems.

Ahkami, Amirhossein↗

Atomic Force Microscopy and Infrared Nanospectroscopy of COVID-19 Spike Protein for the Quantification of Adhesion to Common Surfaces

The COVID-19 pandemic has claimed millions of lives worldwide, sickened many more, and has resulted in severe socio-economic consequences. As society returns to normal, understanding the spread and persistence of SARS CoV-2 on commonplace surfaces can help to mitigate future outbreaks of coronaviruses and other pathogens. We hypothesize that such an understanding can be aided from studying the binding and interaction of viral proteins with non-biological surfaces. Here, we propose a methodology for investigating the adhesion of the SARS CoV-2 spike glycoprotein on common inorganic surfaces such as aluminum-, copper-, iron-, silica-, and ceria-oxides as well as metallic gold. Quantitative adhesion was obtained from the analysis of measured forces at the nanoscale using an atomic force microscope operated under ambient conditions. Without imposing further constraints on the measurement conditions, our preliminary findings suggest that spike glycoproteins interact with similar adhesion forces across the majority of the metal oxides tested with the exception to gold, for which attraction forces ~10 times stronger than all other materials studied were observed. Ferritin, which was used as a reference protein, was found to exhibit similar adhesion forces as SARS CoV-2 spike protein. Further, this study results show that glycoprotein adhesion forces for similar ambient humidity, tip shape, and contact surface are non-specific to the properties of metal oxide surfaces, which are expected to be covered by a thin water film. The findings suggest that at ambient conditions glycoprotein adhesion to metal oxides is primarily controlled by the water capillary forces and they depend on the surface tension of the liquid (water). We discuss further strategies warranted to decipher the intricate nanoscale forces for improved quantification of the adhesion.

59 BASIC BIOLOGICAL SCIENCES↗

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

59 BASIC BIOLOGICAL SCIENCES↗

Framework for idealized climate simulations with spatiotemporal stochastic clouds and planetary-scale circulations

In climate predictions, clouds are the leading source of uncertainty. This is partly because, to simulate the fluid dynamics of climate over the entire globe, a large grid spacing must be used, so clouds are a subgrid-scale parametrization rather than a resolved feature. Here, a framework is investigated with finer grid spacing of O(1) or O(10)km so that some clouds are not subgrid-scale; instead, clouds evolve on the numerical grid. This cloud evolution is achieved using stochastic modeling. Hence, the framework is idealized in the sense that the full fluid dynamics of cloud circulations is still not resolved, and simplified vertical structures are used. Nevertheless, the fluid dynamics model includes evolving clouds that interactively adjust in size, shape, lifetime, and regional coverage. In addition, different cloud types are included with different roles in the climate system, including deep convective clouds and also boundary-layer clouds such as shallow cumulus and stratocumulus clouds. Other basic aspects of the idealized climate system are planetary-scale circulations (e.g., Walker circulation) and radiation. With these ingredients (evolving clouds, planetary-scale circulations, and radiation), the framework has the potential for idealized investigations of climate change with interactive cloud–radiative feedback of individual clouds. Here, the formulation of the model equations is presented, and numerical simulations are shown to illustrate the model dynamics and climate change.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

Signatures of an energetic charged body streaming in a plasma

A charged body moving in a plasma can excite a variety of linear and nonlinear waves in the form of trailing wakes, fore-wake shocks, and precursor solitons. These structures can further interact with the background plasma to create secondary effects that can serve as signatures of the passage of the charged body. Using particle-in-cell simulations, we carry out a basic investigation of the dynamics of a plasma system that is being traversed by an energetic charged body. Using two different shapes of this charged source, namely, an idealized infinite length planar source and a two-dimensional thin rectangular source, we examine the differences in the nature of the excited wave structures and their consequent impact on the background plasma. Our simulations reveal interesting features such as the dependence of the precursor speeds on the total charge on the driving source, local particle trapping, and energization of the trapped particles in various regions along the traversal path leading to the formation of energetic charged beamlets. So our basic findings could find practical applications such as in analyzing the trajectories of charged objects like space debris orbiting in the ionosphere.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Meeting report: The first soil viral workshop 2022

Soil viral ecology is a growing research field; however, the state of knowledge still lags behind that of aquatic systems. Therefore, to facilitate progress, the first Soil Viral Workshop was held to encourage international scientific discussion and collaboration, suggest guidelines for future research, and establish soil viral research as a concrete research area. The workshop took place at Søminestationen, Denmark, between 15 and 17th of June 2022. The meeting was primarily held in person, but the sessions were also streamed online. The workshop was attended by 23 researchers from ten different countries and from a wide range of subfields and career stages. Eleven talks were presented, followed by discussions revolving around three major topics: viral genomics, virus-host interactions, and viruses in the soil food web. The main take-home messages and suggestions from the discussions are summarized in this report.

09 BIOMASS FUELS↗

Thermodynamics and solvent linkage of macromolecule–ligand interactions

Binding involves two steps, desolvation and association. While water is ubiquitous and occurs at high concentration, it is typically ignored. In vitro experiments typically use infinite dilution conditions, while in vivo, the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular milieu. Our paper discusses isothermal titration calorimetry approaches that address the role of water in binding. For example, use of D2O allows the contribution of solvent reorganization to the enthalpy component to be assessed. Furthermore, the addition of osmolytes will decrease the water activity of a solution and allow effects on Ka to be determined. In most cases, binding becomes tighter in the presence of osmolytes as the desolvation penalty associated with binding is minimized. In other cases, the osmolytes prefer to interact with the ligand or protein, and if their removal is more difficult than shedding water, then binding can be weakened. Lastly, these complicating layers can be discerned by different slopes in ln(Ka) vs osmolality plots and by differential scanning calorimetry in the presence of the osmolyte.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structure of frequency-interacting RNA helicase from Neurospora crassa reveals high flexibility in a domain critical for circadian rhythm and RNA surveillance

The FRH (frequency-interacting RNA helicase) protein is the Neurospora crassa homolog of yeast Mtr4, an essential RNA helicase that plays a central role in RNA metabolism as an activator of the nuclear RNA exosome. FRH is also a required component of the circadian clock, mediating protein interactions that result in the rhythmic repression of gene expression. Here we show that FRH unwinds RNA substrates in vitro with a kinetic profile similar to Mtr4, indicating that while FRH has acquired additional functionality, its core helicase function remains intact. In contrast with the earlier FRH structures, a new crystal form of FRH results in an ATP binding site that is undisturbed by crystal contacts and adopts a conformation consistent with nucleotide binding and hydrolysis. Strikingly, this new FRH structure adopts an arch domain conformation that is dramatically altered from previous structures. Comparison of the existing FRH structures reveals conserved hinge points that appear to facilitate arch motion. Regions in the arch have been previously shown to mediate a variety of protein-protein interactions critical for RNA surveillance and circadian clock functions. The conformational changes highlighted in the FRH structures provide a platform for investigating the relationship between arch dynamics and Mtr4/FRH function.

59 BASIC BIOLOGICAL SCIENCES↗

Empirical evidence that glucan-interacting amino acid side chains within the transmembrane channel collectively facilitate cellulose synthase function

The fundamental mechanism of cellulose synthesis is widely conserved across Kingdoms and depends on cellulose synthases, which are processive, dual-function, family 2 glycosyltransferases (GT-2). These enzymes polymerize glucose on the cytoplasmic side of the plasma membrane and export the glucan chain to the cell surface through an integral transmembrane (TM) channel. Structural studies of active plant cellulose synthases (CESAs) have revealed interactions between the nascent glucan chain and the side chains of polar, charged, and aromatic amino acid residues that line the TM channel. However, the functional consequences of modifying these side chains have not been tested in vivo in CESAs or other processive GT-2s. To test this, we used an established in vivo assay based on genetic complementation of CESA5 in the moss, Physcomitrium patens. For accurate prediction of glucan-interacting amino acid residues, we generated a complete homotrimeric molecular model of PpCESA5 using a combination of homology and de novo modeling. All-atom molecular dynamics-based analyses of contact metrics and interaction energy identified 23 amino acid residues with high propensity to interact with the nascent glucan chain within the TM channel or on the apoplastic surface of PpCESA5. Mutating any one of 18 of these amino acid residues to alanine, thereby removing their side chains, abolished or impaired CESA function, with the strongest effects observed upon the loss of charged amino acid side chains. This provides direct evidence to support the hypothesis that multiple amino acid residues collectively maintain a smooth energy landscape within the TM channel to facilitate glucan translocation.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Retractile lysyl-tRNA synthetase-AIMP2 assembly in the human multi-aminoacyl-tRNA synthetase complex

Multi-aminoacyl-tRNA synthetase complex (MSC) is the second largest machinery for protein synthesis in human cells and also regulates multiple nontranslational functions through its components. Prior studies have shown that the MSC can respond to external signals by releasing its components to function outside it. The internal assembly is fundamental to MSC regulation. Here, using crystal structural analyses (at 1.88 Å resolution) along with molecular modeling, gel-filtration chromatography, and co-immunoprecipitation, we report that human lysyl-tRNA synthetase (LysRS) forms a tighter assembly with the scaffold protein aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) than previously observed. Wefound that two AIMP2 N-terminal peptides form an antiparallel scaffold and hold two LysRS dimers through four binding motifs and additional interactions. Of note, the four catalytic subunits of LysRS in the tightly assembled complex were all accessible for tRNA recognition. We further noted that two recently reported human disease-associated mutations conflict with this tighter assembly, cause LysRS release from the MSC, and inactivate the enzyme. These findings reveal a previously unknown dimension of MSC subcomplex assembly and suggest that the retractility of this complex may be critical for its physiological functions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

From Protein Design to the Energy Landscape of a Cold Unfolding Protein

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein–water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ~0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

59 BASIC BIOLOGICAL SCIENCES↗

Synthesis, crystal structure, and magnetic properties of bis(aqua)[μ-(terepthalato-κo,κo´)]copper(II)monohydrate [Cu(C₈O₄)(OH₂)₂]·H₂O

A novel one dimensional Cu(II) coordination polymer [Cu(C₈O₄)(OH₂)₂] ·H₂O 1 has been synthesized and characterized by elemental analysis, IR spectroscopy, X-ray crystal diffraction and magnetic measurements. Single-crystal structural analysis shows that complex 1 consist of 1D Cu(II) chain containing threefold bridging ligands, one (syn–syn) carboxylate and two water molecules. The magnetic study of 1 has been investigated, indicating an overall antiferromagnetic interaction in the complex. - Graphical abstract: The complex [Cu(C₈O₄)(OH₂)₂] ·H₂O was prepared by the reaction of solid copper(II)basic carbonate with an aqueous solution of sodium terephthalate under heating at 50–60 {sup o}C after which it was allowed to crystallize at room temperature. The structure of the complex is distorted octahedral. Magnetic study of the complex reveals antiferromagnetic behaviour. Highlights: • A novel copper(II) coordination polymer with terepthalate dianion was grown in an aqueous solution. • The single crystal growth temperature was 25 °C for period of two weeks. • The magnetic property of the complex was studied using SQUID. • The complex showed antiferromagnetic properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Climate change alters slug abundance but not herbivory in a temperate grassland

Climate change will significantly impact the world’s ecosystems, in part by altering species interactions and ecological processes, such as herbivory and plant community dynamics, which may impact forage quality and ecosystem production. Yet relatively few field experimental manipulations assessing all of these parameters have been performed to date. To help fill this knowledge gap, we evaluated the effects of increased temperature (+3°C day and night, year-round) and precipitation (+30% of mean annual rainfall) on slug herbivory and abundance and plant community dynamics biweekly in a pasture located in central Kentucky, U.S.A. Warming increased slug abundance once during the winter, likely due to improving conditions for foraging, whereas warming reduced slug abundance at times in late spring, mid-summer, and early fall (from 62–95% reduction depending on month). We found that warming and increased precipitation did not significantly modify slug herbivory at our site, despite altering slug abundance and affecting plant community composition and forage quality. Climate change will alter seasonal patterns of slug abundance through both direct effects on slug biology and indirect effects mediated by changes in the plant community, suggesting that pasture management practices may have to adapt.

59 BASIC BIOLOGICAL SCIENCES↗