19Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for 20 improving agricultural crop productivity. Here we reveal high diversity in the kinetic 21 response between 10°C to 37°C by Rubisco from C3-and C4-species within the grass tribe 22Paniceae. The CO2-fixation rate (kcat C ) for Rubisco from the C4-grasses with NADP-malic 23 enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic 24 pathways was two-fold greater than the kcat C of Rubisco from NAD-ME species over all 25 temperatures. The decline in the response of CO2/O2 specificity with increasing 26 temperature was slower for PCK and NADP-ME Rubisco -a trait which would be 27 advantageous in the warmer climates they inhabit relative to the NAD-ME grasses. 28Variation in the temperatures kinetics of Paniceae C3-Rubisco and PCK-Rubisco were 29 modelled to differentially stimulate C3-photosynthesis above and below 25°C under current 30 and elevated CO2. Identified are large subunit amino acid substitutions that could account 31 for the catalytic variation among Paniceae Rubisco. Incompatibilities with Paniceae 32Rubisco biogenesis in tobacco however hindered their mutagenic testing by chloroplast 33 transformation. Circumventing these bioengineering limitations is critical to tailoring the 34 properties of crop Rubisco to suit future climates. 35Concerns about how escalating climate change will influence ecosystems are particularly 36 focused on the consequences to global agricultural productivity where increases are 37 paramount to meet the rising food and biofuel demands. Strategies to improve crop yield 38 by increasing photosynthesis have largely focused on overcoming the functional 39 inadequacies of the CO2-fixing enzyme Rubisco. A competing O2-fixing reaction by 40Rubisco produces a toxic product whose recycling by photorespiration consumes energy 41 and releases carbon. The frequency of the oxygenation reaction increases with temperature. 42To evade photorespiration many plants from hot, arid ecosystems have evolved C4 43 photosynthesis that concentrates CO2 around Rubisco that also facilitates improved plant 44water, light and nitrogen use. Here we show extensive catalytic variation in Rubisco from 45Paniceae grasses that align with the biochemistry and environmental origins of the different 46 C4 plant subtypes. We reveal opportunities for enhancing crop photosynthesis under 47 current and future CO2 levels at varied temperatures. 48The realization of the dire need to address global food security has heightened the need for 49 new solutions to increase crop yields 1 . Field tests and modelling analyses have highlighted 50 how photosynthetic carbon assimilation underpins the maximal yield potential of crops 2 . 51This has increased efforts to identify solutions to enhance photosynthetic efficiency and 52 hence plant productivity 3 . Particular attention is being paid to improving the rate at which 53 ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) can fix CO2 54 (refs...

Omono River virus (OmRV) is a double-stranded RNA virus isolated from Culex mosquitos, and it belongs to a group of unassigned insect viruses that appear to be related to Totiviridae. This paper describes electron cryo-microscopy (cryoEM) structures for the intact OmRV virion to 8.9 Å resolution and the structure of the empty virus-like-particle, that lacks RNA, to 8.3 Å resolution. The icosahedral capsid contains 120-subunits and resembles another closely related arthropod-borne totivirus-like virus, the infectious myonecrosis virus (IMNV) from shrimps. Both viruses have an elevated plateau around their icosahedral 5-fold axes, surrounded by a deep canyon. Sequence and structural analysis suggests that this plateau region is mainly composed of the extended C-terminal region of the capsid proteins. In contrast to IMNV, the infectious form of OmRV lacks extensive fibre complexes at its 5-fold axes as directly confirmed by a contrast-enhancement technique, using Zernike phase-contrast cryo-EM. Instead, these fibre complexes are replaced by a short “plug” structure at the five-fold axes of OmRV. OmRV and IMNV have acquired an extracellular phase, and the structures at the five-fold axes may be significant in adaptation to cell-to-cell transmission in metazoan hosts.

The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)—a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhanced RsRubisco activation to 75% under elevated CO2 (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2 specificity impaired 30–50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.