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Carboxysomes are protein microcompartments found in cyanobacteria, whose shell encapsulates rubisco at the heart of carbon fixation in the Calvin cycle. Carboxysomes are thought to locally concentrate CO₂in the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, permeability coefficients have not previously been determined for these gases, or for Calvin-cycle intermediates such as bicarbonate ( ${\text{HCO}}_3^{-}$), 3-phosphoglycerate, or ribulose-1,5-bisphosphate. Starting from a high-resolution cryogenic electron microscopy structure of a synthetic $\beta$-carboxysome shell, we perform unbiased all-atom molecular dynamics to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the bacterial microcompartment trimer proteins and encapsulation peptides, is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to ${\text{HCO}}_3^{-}$relative to CO₂. To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the CO₂-concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics simulations estimate the mean first passage time for CO₂assimilation in a crowded model carboxysome. Despite a relatively high CO₂permeability of approximately 10⁻²cm/s across the carboxysome shell, the shell proteins reflect enough CO₂back toward rubisco that 2,650 CO₂molecules can be fixed by rubisco for every 1 CO₂molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications. 

Abstract Antifungal echinocandins inhibit the biosynthesis of β−1,3-glucan, a major and essential polysaccharide component of the fungal cell wall. However, the efficacy of echinocandins against the pathogenAspergillus fumigatusis limited. Here, we use solid-state nuclear magnetic resonance (ssNMR) and other techniques to show that echinocandins induce dynamic changes in the assembly of mobile and rigid polymers within theA. fumigatuscell wall. The reduction of β−1,3-glucan induced by echinocandins is accompanied by a concurrent increase in levels of chitin, chitosan, and highly polymorphic α−1,3-glucans, whose physical association with chitin maintains cell wall integrity and modulates water permeability. The rearrangement of the macromolecular network is dynamic and controls the permeability and circulation of the drug throughout the cell wall. Thus, our results indicate that echinocandin treatment triggers compensatory rearrangements in the cell wall that may helpA. fumigatusto tolerate the drugs’ antifungal effects. 

Single particle analysis cryo-electron microscopy (EM) and molecular dynamics (MD) have been complimentary methods since cryo-EM was first applied to the field of structural biology. The relationship started by biasing structural models to fit low-resolution cryo-EM maps of large macromolecular complexes not amenable to crystallization. The connection between cryo-EM and MD evolved as cryo-EM maps improved in resolution, allowing advanced sampling algorithms to simultaneously refine backbone and sidechains. Moving beyond a single static snapshot, modern inferencing approaches integrate cryo-EM and MD to generate structural ensembles from cryo-EM map data or directly from the particle images themselves. We summarize the recent history of MD innovations in the area of cryo-EM modeling. The merits for the myriad of MD based cryo-EM modeling methods are discussed, as well as, the discoveries that were made possible by the integration of molecular modeling with cryo-EM. Lastly, current challenges and potential opportunities are reviewed. 

Summary As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic algaCyanidioschyzon merolaewas more thermotolerant than those from mesophilic plants, includingArabidopsis thaliana. To understand enzyme features underlying the thermotolerance ofC. merolaeGLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold‐predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar K_mfor ATP compared to AtGLYK, its K_mfor glycerate increased threefold. This study demonstrates that molecular dynamics simulation‐guided structure‐based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.