Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesi
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Dietz, Jonathan V.↗
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Ground Heat Flux Reconstruction Using Bayesian Uncertainty Quantification Machinery and Surrogate Modeling
Ground heat flux (G 0 ) is a key component of the land-surface energy balance of high-latitude regions. Despite its crucial role in controlling permafrost degradation due to global warming, G 0 is sparsely measured and not well represented in the outputs of global scale model simulation. In this study, an analytical heat transfer model is tested to reconstruct G 0 across seasons using soil temperature series from field measurements, Global Climate Model, and climate reanalysis outputs. The probability density functions of ground heat flux and of model parameters are inferred using available G 0 data (measured or modeled) for snow-free period as a reference. When observed G 0 is not available, a numerical model is applied using estimates of surface heat flux (dependent on parameters) as the top boundary condition. These estimates (and thus the corresponding parameters) are verified by comparing the distributions of simulated and measured soil temperature at several depths. Aided by state-of-the-art uncertainty quantification methods, the developed G 0 reconstruction approach provides novel means for assessing the probabilistic structure of the ground heat flux for regional permafrost change studies.
54 ENVIRONMENTAL SCIENCES↗
RIViT-seq enables systematic identification of regulons of transcriptional machineries
Abstract Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcription factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is laborious and not scalable. Here, we demonstrate regulon identification by in vitro transcription-sequencing (RIViT-seq) that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViT-seq data expands the transcriptional regulatory network in this bacterium, discovering regulatory cascades and crosstalk between sigma factors. Implementation of RIViT-seq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.
59 BASIC BIOLOGICAL SCIENCES↗
Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery
Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.
59 BASIC BIOLOGICAL SCIENCES↗
Molecular model of TFIIH recruitment to the transcription-coupled repair machinery
Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4CSA ubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR.
Paul, Tanmoy↗
Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins
Radiation of the plant pyridoxal 5'-phosphate (PLP)-dependent aromatic L -amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L -amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. In this paper, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.
59 BASIC BIOLOGICAL SCIENCES↗
Comparative Genomic Analyses of Cellulolytic Machinery Reveal Two Nutritional Strategies of Marine Labyrinthulomycetes Protists
Labyrinthulomycetes are a group of ubiquitous and diverse unicellular Stramenopiles and have long been known for their vital role in ocean carbon cycling. However, their ecological function from the perspective of organic matter degradation remains poorly understood. This study reports high-quality genomes of two newly isolated Labyrinthulomycetes strains, namely, Botryochytrium sp. strain S-28 and Oblongichytrium sp. strain S-429, and provides molecular analysis of their ecological functions using comparative genomics and a biochemical assay. Our results suggest that Labyrinthulomycetes may occupy multiple ecological niches in marine ecosystems because of the significant differences in gene function among different genera. Certain strains could degrade wheat bran independently by secreting cellulase. The key glycoside hydrolase families (GH1, GH5, and GH9) related to cellulase and the functional domains of carbohydrate-active enzymes (CAZymes) were more enriched in their genomes. This group can actively participate in marine biochemical cycles as decomposers. In contrast, other strains that could not produce cellulase may thrive as “leftover scavengers” and act as a source of nutrients to the higher-trophic-level plankton. In addition, our findings emphasize the dual roles of endoglucanase, acting as both exo- and endoglucanases, in the process of cellulose degradation. Using genomic, biochemical, and phylogenetic analyses, our study provides a broader insight into the nutritional patterns and ecological functions of Labyrinthulomycetes.
59 BASIC BIOLOGICAL SCIENCES↗
Advanced Airfoils for Efficient Combined Heat Power Systems: Task 4.3 - Gas Turbine Machinery and Systems
Industrial gas turbines are commonly used in steam combined heat and power (CHP) applications. CHP applications have significant environmental and economic benefits that are consistent with the goals of the U.S. Department of Energy. This presentation provides a status update for a DOE effort investigating the impacts of advanced internal cooling technologies for small (5-10 MW) gas turbine CHP applications. The potential efficiency impacts are 2-3 percentage points based on the model and the cooling technologies investigated in this project.
Straub, Douglas↗
Device for controlling additive manufacturing machinery
A computing device for controlling the operation of an additive manufacturing machine comprises a memory element and a processing element. The memory element is configured to store a three-dimensional model of a part to be manufactured, wherein the three-dimensional model defines a plurality of cross sections of the part. The processing element is in communication with the memory element. The processing element is configured to receive the three-dimensional model, determine a plurality of paths, each path including a plurality of parallel lines, determine a radiation beam power for each line, such that the radiation beam power varies non-linearly according to a length of the line, and determine a radiation beam scan speed for each line, such that the radiation beam scan speed is a function of a temperature of a material used to manufacture the part, the length of the line, and the radiation beam power for the line.
Barr, Christian↗
Device for controlling additive manufacturing machinery
A computing device for controlling the operation of an additive manufacturing machine comprises a memory element and a processing element. The memory element is configured to store a three-dimensional model of a part to be manufactured, wherein the three-dimensional model defines a plurality of cross sections of the part. The processing element is in communication with the memory element. The processing element is configured to receive the three-dimensional model, determine a path across a surface of each cross section, wherein the path includes a plurality of parallel lines, calculate a power for a radiation beam to scan each of the lines, such that the power varies from line to line non-linearly according to a length of the line, and calculate a scan speed for the radiation beam for each of the lines, such that the scan speed varies line to line non-linearly according to the power of the radiation beam.
Barr, Christian↗
Device for controlling additive manufacturing machinery
A computing device for controlling the operation of an additive manufacturing machine comprises a memory element and a processing element. The memory element is configured to store a three-dimensional model of a part to be manufactured, wherein the three-dimensional model defines a plurality of cross sections of the part. The processing element is in communication with the memory element. The processing element is configured to receive the three-dimensional model, determine a plurality of paths, each path including a plurality of parallel lines, determine a radiation beam power for each line, such that the radiation beam power varies non-linearly according to a length of the line, and determine a radiation beam scan speed for each line, such that the radiation beam scan speed is a function of a temperature of a material used to manufacture the part, the length of the line, and the radiation beam power for the line.
Barr, Christian↗
Paper #50 Preliminary Sizing of Active Magnetic Bearings for sCO2 Waste-Heat Recovery Application
The team of Southwest Research Institute (SwRI) and GE Vernova (GE) is executing a project developing conceptual designs of high-temperature active magnetic bearings (AMBs) for sCO2 machinery applications. The goals of the project are to develop conceptual designs for radial and thrust AMBs capable of operating in sCO2 environments up to 1000°F. A waste-heat recovery (WHR) application with hermetic machinery is used as a reference case to apply AMBs. This application was previously investigated to compare hermetic machinery enabled by process-lubricated bearings and high-speed motors/generators with traditional machinery configurations using oil-lubricated bearings, gearboxes, and grid-frequency electric machines. In this paper, AMBs are sized for the WHR application machinery. Load capacity estimates are made using first-principle-derived formulas that consider operation at elevated temperatures. Estimates of linearized AMB system coefficients are also made to aid future controller development. Rotor models are developed that account for AMB rotor geometry and housing space requirements. Rotordynamics studies are performed to identify reasonable target values for closed-loop AMB stiffness and closed-loop AMB damping. These target values will be used in future control studies.
Lipham, Robert↗
Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts
Significance Chloroplasts are of vital importance in photosynthetic eukaryotic organisms. Like mitochondria, they contain their own genomes. Nevertheless, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and must cross the chloroplast envelope membranes to reach their proper destinations inside the organelle. Despite its fundamental role, our knowledge of the machinery catalyzing chloroplast protein import is incomplete and controversial. Here, we address the evolutionary conservation, composition, and function of the chloroplast protein import machinery using the green alga Chlamydomonas reinhardtii as a model system. Our findings help clarify the current debate regarding the composition of the chloroplast protein import machinery, provide evidence for cross-compartmental coordination of its biogenesis, and open promising avenues for its structural characterization.
54 ENVIRONMENTAL SCIENCES↗
Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum
Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.
59 BASIC BIOLOGICAL SCIENCES↗
Nanoscale resolution of microbial fiber degradation in action
The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical Clostridium thermocellum system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form ‘cellulosome capsules’ driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.
59 BASIC BIOLOGICAL SCIENCES↗
Designing Harvesting and Hauling Cost Models for Energy Cane Production for Biorefineries
The harvesting and hauling operations of bioenergy feedstock is an important area in biofuel production. Production costs can be minimized by maintaining optimal machinery units for these operations. The objective of this study is to design an optimal harvesting unit for bioenergy refinery and estimate harvesting and hauling costs of energy cane. A biorefinery with the annual capacity of processing twenty-five million imp. gallons of ethanol were considered. Given the efficiency of harvesting, a two-row soldier system was considered. Considering the year-round supply of energy cane to the refinery, the optimal machinery unit was designed, and the combined operation costs were derived. The average estimated ownership, repair, labor and fuel and lubricant costs of biomass harvest unit were calculated to be $\$$0.50, $\$$0.54, $\$$1.78 and $\$$1.51/mt, respectively. The costs distribution generated showed harvesting and hauling costs could range between $\$$5.47–$\$$9.23/mt of energy cane. The methodology and the research output will provide guidelines for investors in designing harvesting and hauling units and estimating costs for different scales of operation.
09 BIOMASS FUELS↗
Heavy-Duty Nonroad Material Handler Electrification Part 2: Energy Efficiency and Performance Evaluation
Decarbonization efforts achieved through electrification in nonroad mobile machinery can realize a reduction in fuel consumption of more than 20%, thanks to concepts familiar to light-duty passenger vehicles. This case study compares the results of a hybrid-electric material handler to its conventional counterpart, utilizing machine-specific drive cycles presented in part one of this paper series. The hybrid prototype features an extended-range electric vehicle (EREV) powertrain that demonstrated substantial energy efficiency improvements. Specifically, there was a reduction in equivalent fuel consumption of 75% when operating in electric-only mode, and 33% when maintaining the battery by charging with an on-board generator. Together, the efficiency improvements can be extrapolated over a low-intensity, 8-h shift characterized by significant idle time and highly dynamic engine load for a 47% reduction in net energy consumption. Key technologies that led to this improvement included engine downsizing and decoupling, regenerative braking, and an electrohydraulic pump unit with advanced controls. Finally, his study explains details of the powertrain architecture and subsystems that were implemented on a demonstration vehicle, control strategies used to meet project goals, and an analysis of energy consumption from testing on a closed course. Also included in this study is a discourse on comparison metrics that can be used for quantifying the energy consumption differences between hybrid-electric and conventional diesel powertrains in nonroad mobile machinery.
25 ENERGY STORAGE↗