Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of ...gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe the use of a chemical landing matrix that preserves the structural integrity of the deposited particles. With this system we obtain a three-dimensional reconstruction of the 800 kDa protein complex GroEL from gas-phase deposited GroEL ions. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.
Ecologists have studied the role of interspecific competition in structuring ecological communities for decades. Differential weather effects on animal competitors may be a particularly important ...factor contributing to the outcome of competitive interactions, though few studies have tested this hypothesis in free‐ranging animals. Specifically, weather might influence competitive dynamics by altering competitor densities and/or per‐capita competitive effects on demographic vital rates. We used a 9‐year data set of marked individuals to test for direct and interactive effects of weather and competitor density on survival probability in two coexisting mammalian congeners: Columbian ground squirrels (Urocitellus columbianus) and northern Idaho ground squirrels (Urocitellus brunneus). Ambient temperature and precipitation influenced survival probability in both species, but the effects of weather differed between the two species. Moreover, density of the larger Columbian ground squirrel negatively impacted survival probability in the smaller northern Idaho ground squirrel (but not vice versa), and the strength of the negative effect was exacerbated by precipitation. That is, cooler, wetter conditions benefited the larger competitor to the detriment of the smaller species. Our results suggest weather‐driven environmental variation influences the competitive equilibrium between ecologically similar mammals of differential body size. Whether future climate change leads to the competitive exclusion of either species will likely depend on the mechanism(s) explaining the coexistence of these competing species. Divergent body size and, hence, differences in thermal tolerance and giving up densities offer potential explanations for the weather‐dependent competitive asymmetry we documented, especially if the larger species competitively excludes the smaller species from habitat patches of shared preference via interference.
Food acquisition is among the most important tasks faced by free-ranging animals. Predation and thermal risks, however, can make foraging a costly endeavor and foraging can preclude other important ...activities. Moreover, seasonal life cycle events such as hibernation impose energetic thresholds and time constraints on foraging. These factors interact with an animal’s endogenous state to influence foraging behavior. We tested a suite of predictions based on foraging theory to explore the effects of thermal environment, body condition, and conspecific density on aboveground activity (which is primarily foraging activity) of the northern Idaho ground squirrel (
Urocitellus brunneus
), an imperiled rodent that hibernates for 9 months each year. We took advantage of the squirrels’ semi-fossorial lifestyle to document daily aboveground activity by attaching geolocators to squirrels. We modeled squirrel activity with generalized linear mixed-effects models to document the relative importance of thermal environment, body condition, and conspecific density for daily aboveground activity. Aboveground activity by northern Idaho ground squirrels increased throughout their active season and leaner squirrels increased their activity more than heavier squirrels as residual foraging opportunities diminished. Thermal conditions also influenced squirrel activity: squirrels spent less time above ground during extreme temperatures and on days with significant precipitation. Aboveground activity of northern Idaho ground squirrels largely adhered to predictions of risk-sensitive and state-dependent foraging theory. Management actions that enhance forage will likely improve the probability of recovery for this federally threatened species by minimizing trade-offs squirrels need to make to acquire sufficient food to survive hibernation and reproduce in subsequent years.
Significance statement
Acquiring food is a vital task for wild animals, but foraging can be dangerous. Hibernation imposes annual energetic requirements animals must meet within a short time when food is available and the animal is active. Hibernating species, therefore, must navigate trade-offs among foraging, predation risk, and thermal intolerance. We investigated how these pressures influence daily foraging activity of the northern Idaho ground squirrel (
Urocitellus brunneus
), a federally threatened species. Ground squirrels forage above ground during the active season and retreat to burrows to avoid predation and extreme weather but accept greater risks to forage as hibernation approaches. Additionally, lean squirrels with high energetic needs forage more than heavy squirrels, exposing lean squirrels to higher predation risk. Improving forage may improve recovery odds for this imperiled species by allowing squirrels to reduce their mortality risk.
Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing ...matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexes─GroEL, the 20S proteasome core particle, and β-galactosidase─and obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (∼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.
Hibernation is a remarkable behaviour deployed by a diverse array of endotherms within many clades that greatly reduces metabolic need, but also has somatic costs. Hibernation in modern endotherms is ...often assumed to be an adaptation allowing animals to avoid extreme thermal conditions or food shortages in seasonal environments. However, many animals hibernate when foraging conditions are energetically profitable, suggesting other causal factors influence hibernation behaviour.
Understanding the selection pressures responsible for intraspecific variation in the timing and duration of hibernation can help elucidate the relative evolutionary influences of the ultimate ecological causes of hibernation. We tested four previously proposed mechanistic hypotheses to explain intraspecific variation in hibernation phenology in the federally threatened northern Idaho ground squirrel (Urocitellus brunneus): (1) thermal tolerance, (2) food limitation, (3) predation avoidance and (4) sexual selection.
The predation avoidance and sexual selection hypotheses received the most support, although we also found some support for the thermal tolerance and food limitation hypotheses. Heavy squirrels increased hibernation duration regardless of environmental conditions, as predicted solely by the predation avoidance hypothesis. Reproductive males emerged from hibernation earlier in spring than other sex–age classes, a pattern predicted by the sexual selection hypothesis. Temperature and food availability explained a much smaller amount of the variation in hibernation behaviour, only partially supporting predictions of the thermal tolerance and food limitation hypotheses.
Our results indicate that animals navigate life‐history trade‐offs between energetic allocation to survival and reproduction via state‐dependent optimization of hibernation phenology. Consequently, any future environmental changes that influence body condition will have implications for population ecology and life‐history evolution of hibernating animals due to stark differences in daily survival probability between hibernation and the active season.
Read the free Plain Language Summary for this article on the Journal blog.
Read the free Plain Language Summary for this article on the Journal blog.
Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the ...plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air–water interface often leads to sample damage and preferential orientation of the particles. Using native mass spectrometry to create cryoEM samples, potentially avoids these problems and allows the use of mass spectrometry sample isolation techniques during EM grid creation. We review the recent publications that have demonstrated protein complexes can be ionized, flown through the mass spectrometer, gently landed onto EM grids, imaged, and reconstructed in 3D. Although many uncertainties and challenges remain, the combination of cryoEM and MS has great potential.
Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools ...for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.
Riboflavin receptor 3 (RFVT3) is a key protein in energetic metabolism reprogramming and is overexpressed in multiple cancers involved in malignant proliferation, angiogenesis, chemotherapy ...resistance, and immunosuppression. To enable non-invasive real-time quantification of RFVT3 in tumors, we sought to develop a suitable PET probe that would allow specific and selective RFVT3 imaging in vivo. A novel radiofluorinated riboflavin probe (18F-RFTA) based on riboflavin was synthesized and characterized in terms of radiochemical purity, hydrophilicity, binding affinity, and stability. Positron emission tomography (PET) imaging of 18F-RFTA was performed in U87MG tumor-bearing mice. Immunohistochemistry staining was carried out to determine the expression of RFVT3 in U87MG tumors. 18F-RFTA was characterized by high radiochemical purity and RFVT3 binding affinity, and remarkable stability in vitro and in vivo. Small-animal PET imaging with 18F-RFTA revealed significantly higher uptake in RFVT3-expressing U87MG tumors than in muscle. In conclusion, we have developed the first radiofluorinated riboflavin-based PET probe that is suitable for imaging RFVT3-positive tumors. The new target/probe system can be leveraged for extensive use in the diagnosis and treatment of RFVT3 overexpressing diseases, such as oncologic, cardiovascular, and neurodegenerative diseases.
Display omitted
•A novel PET probe is designed and developed to achieve riboflavin receptor-targeted imaging of glioma mouse models.•The probe exhibited promising pharmacological properties for future translation studies.•The new target/probe system can be leveraged for use in the diagnosis and treatment of riboflavin receptor-related diseases.
•Realised ES are a product of the potential service and specified beneficiaries.•Natural capital (NC) and human-derived capital (HDC) are both essential for ES.•HDC plays a role even at the stage of ...potential ecosystem services.•It is possible but not always easy to separate the contribution of NC and HDC to ES.•Sustainable management should identify critical NC and HDC for each service.
Display omitted
There is growing interest in the role that natural capital plays in underpinning ecosystem services. Yet, there remain differences and inconsistencies in the conceptualisation of capital and ecosystem services and the role that humans play in their delivery. Using worked examples in a stocks and flows systems approach, we show that both natural capital (NC) and human-derived (produced, human, social, cultural, financial) capital (HDC) are necessary to create ecosystem services at many levels. HDC plays a role at three stages of ecosystem service delivery. Firstly, as essential elements of a combined social-ecological system to create a potential ecosystem service. Secondly, through the beneficiaries in shaping the demand for that service. Thirdly, in the form of additional capital required to realise the ecosystem service flow. We show that it is possible, although not always easy, to separately identify how these forms of capital contribute to ecosystem service flow. We discuss how applying a systems approach can help identify critical natural capital and critical human-derived capital to guide sustainable management of the stocks and flows of all forms of capital which underpin provision of multiple ecosystem services. The amount of realised ecosystem service can be managed in several ways: via the NC & HDC which govern the potential service, and via factors which govern both the demand from the beneficiaries, and the efficiency of use of the potential service by those beneficiaries.
PDF