Ros, A. | Search bioRxiv

bioRxiv2024

Minimized Sample Consumption for Time-Resolved Serial Crystallography Applied to the Redox Cycle of Human NQO1

Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme.

biochemistry↗

bioRxiv2022

Rapid intralacustrine evolution of an invasive pelagic three-spined stickleback (Gasterosteus aculeatus) ecotype in Lake Constance.

The rapid invasion of the pelagic zone in Lake Constance by three-spined sticklebacks (Gasterosteus aculeatus) since 2012 and their subsequent drastic population growth has had stark ecosystem-wide effects, such as food-web shifts and declines in native biodiversity, including commercially important fish species. Yet, the origin of this invasive pelagic ecotype remains unclear. This study aims to determine if the pelagic ecotype arose in situ from the existing littoral population or following external colonisation, identify potential phenotypic differences between individuals from different habitats, and assess genomic signals of selection. Integrating RAD-sequencing of Lake Constance individuals and whole-genome sequence data for European outgroup populations, this study shows that the pelagic Lake Constance population likely arose recently within the lake from the littoral population, with only weak genome-wide differentiation between individuals from different habitats. This is further supported by minimal differences in meristic and morphometric traits, with shape differences only found between pelagic/inflow sticklebacks and littoral sticklebacks. Using genome scans, we identified multiple outlier loci between littoral and pelagic ecotypes across the genome, potentially suggesting early signs of sympatric speciation despite high connectivity. Furthermore, increased differentiation between pelagic and littoral sticklebacks for body shape-associated loci and the overlap of outlier loci with quantitative trait loci for ecologically relevant traits points toward a driving role of selection in this pelagic invasion. This study provides an important example of rapid ecological diversification from standing genetic variation and a rare case of littoral-pelagic ecotype divergence under high gene flow in a large oligotrophic lake. Ultimately, the results of this study will have major implications for the management of the invasive pelagic ecotype, and the entire stickleback population as a whole.

evolutionary biology↗