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Atlantic ghost crabs (Ocypode quadrata) are predators of beach-nesting shorebird nests and chicks on the United States’ Atlantic and Gulf coasts. Ghost crabs may also disturb birds, altering foraging, habitat use, or nest and brood attendance patterns. Shorebird conservation strategies often involve predator and disturbance management to improve reproductive success, but efforts rarely target ghost crabs. Despite the threat to shorebird reproductive success, ghost crabs are a poorly understood part of the beach ecosystem and additional knowledge about ghost crab habitat selection is needed to inform shorebird conservation. We monitored ghost crab activity, defined as burrow abundance, throughout the shorebird breeding season on Metompkin Island, Virginia, an important breeding site for piping plovers (Charadrius melodus) and American oystercatchers (Haematopus palliatus). We counted burrows at shorebird nests and random locations throughout the breeding season and investigated whether ghost crab activity was greater at nest sites relative to random locations without shorebird nests. While we observed burrows at all nest sites (n= 63 nests), we found that burrow counts were lower at piping plover nests with shell cover, relative to random locations with no shell cover. Ghost crabs may avoid piping plover nest sites due to anti-predator behaviors from incubating adults or differences in microhabitat characteristics selected by piping plovers. We also investigated the effects of habitat type, date, and air temperature on the abundance of ghost crab burrows. We found that while crab burrows were present across the barrier island landscape, there were more burrows in sandy, undisturbed habitats behind the dunes, relative to wave-disturbed beach. Additionally, ghost crab activity increased later in the shorebird breeding season. Understanding when and where ghost crabs are most likely to be active in the landscape can aid decision-making to benefit imperiled shorebird populations. 

Abstract Insertions and deletions (indels) enable evolution and cause disease. Due to technical challenges, indels are left out of most mutational scans, limiting our understanding of them in disease, biology, and evolution. We develop a low cost and bias method, DIMPLE, for systematically generating deletions, insertions, and missense mutations in genes, which we test on a range of targets, including Kir2.1. We use DIMPLE to study how indels impact potassium channel structure, disease, and evolution. We find deletions are most disruptive overall, beta sheets are most sensitive to indels, and flexible loops are sensitive to deletions yet tolerate insertions. 

Understanding factors that influence a species’ distribution and abundance across the annual cycle is required for range-wide conservation. Thousands of imperiled red knots ( Calidris cantus rufa ) stop on Virginia’s barrier islands each year to replenish fat during spring migration. We investigated the variation in red knot presence and flock size, the effects of prey on this variation, and factors influencing prey abundance on Virginia’s barrier islands. We counted red knots and collected potential prey samples at randomly selected sites from 2007–2018 during a two-week period during early and peak migration. Core samples contained crustaceans (Orders Amphipoda and Calanoida), blue mussels ( Mytilus edulis) , coquina clams ( Donax variabilis ), and miscellaneous prey (horseshoe crab eggs ( Limulus polyphemus ), angel wing clams ( Cyrtopleura costata ), and other organisms (e.g., insect larvae, snails, worms)). Estimated red knot peak counts in Virginia during 21–27 May were highest in 2012 (11,959) and lowest in 2014 (2,857; 12-year peak migration x ¯ = 7,175, SD = 2,869). Red knot and prey numbers varied across sampling periods and substrates (i.e., peat and sand). Red knots generally used sites with more prey. Miscellaneous prey ( x ¯ = 2401.00/m 2 , SE = 169.16) influenced red knot presence at a site early in migration, when we only sampled on peat banks. Coquina clams ( x ¯ = 1383.54/m 2 , SE = 125.32) and blue mussels ( x ¯ = 777.91/m 2 , SE = 259.31) affected red knot presence at a site during peak migration, when we sampled both substrates. Few relationships between prey and red knot flock size existed, suggesting that other unmeasured factors determined red knot numbers at occupied sites. Tide and mean daily water temperature affected prey abundance. Maximizing the diversity, availability, and abundance of prey for red knots on barrier islands requires management that encourages the presence of both sand and peat bank intertidal habitats. 

Shorebird reproductive success monitoring often relies on surveys of nest and brood survival. However, conclusions may be inaccurate due to the challenges of gathering and interpreting evidence of nest and brood fate. We tested the efficacy of in-person versus camera- based monitoring to quantify productivity and evaluate threats to reproductive success of American Oystercatchers (Haematopus palliatus) and Piping Plovers (Charadrius melodus) at Metompkin Island, Virginia. We deployed 73 cameras using three set-ups: at nests, at brood sites, and along a transect. The same areas were also surveyed in-person approximately once per week. Camera monitoring confirmed nest fate where in-person monitors could not determine fate from field evidence and provided insight to the effectiveness of mammalian predator removal. However, cameras failed to capture causes of mortality for mobile chicks and did not consistently document chicks where in-person monitoring confirmed successful broods. Cameras produced large quantities of data requiring 63.5–315 hours to review, depending on camera set- up. We found cameras were useful for validating conclusions from in-person monitoring, highlighting threats that surveys missed, and characterizing the predator community. Managers should consider the tradeoff between potential benefits and required effort of camera monitoring when deciding which method would be effective for meeting management goals. 

Abstract Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics. 

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k_cat/K_M146 M⁻¹s⁻¹). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k_cat/K_M103,000 M⁻¹s⁻¹) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. 

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data forPseudomonas putidaketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.