Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization

Quantifying the stabilizing effects of protein–ligand interactions in the gas phase

The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means

Root biology never sleeps

11th Symposium of the International Society of Root Research (ISRR11) and the 9th International Symposium on Root Development (Rooting2021), 24–28 May 2021. Emerging frontiers: root and rhizosphere research in the context of global environmental change. Natural ecosystems and agricultural production have been threatened by multifaceted global environmental changes. Soil degradation, extreme drought and flooding events, shifting climatic patterns and other challenges have prompted many disciplines within plant science to pivot to find solutions. Accordingly, root research has expanded from fundamental studies on roots, as providers of physical support, water, and essential nutrients uptake, towards identification of beneficial traits for stress adaptation and control of key biological soil processes. Advances in trait identification, data acquisition, management and modelling are enabling root researchers to develop predictive models to support ecosystems in these changing environments. Through technical presentations, posters, industry exhibits and a root phenotyping workshop, the international, jointly presented, completely virtual International Society of Root Research (ISRR)11/Rooting2021 meeting provided a unique platform for researchers across disciplines to share recent advances in root biology, from molecular to ecosystem-level scales, in agricultural and natural ecosystems, addressing critical questions in response to climate change and its impact on crop productivity and ecosystem services. In this report, the 2021 ISRR Ambassador cohort provides an overview of the current root research landscape and reflection on the importance of frontier research for a more sustainable future