VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Hydrothermal Synthesis of Monoclinic VO2 Microparticles without Use of Hazardous Reagents: A Key Role for the W-Dopant.

Monoclinic vanadium dioxide (VO2 (M)) is a promising material for various applications ranging from sensing to signature management and smart windows. Most applications rely on its reversible structural phase transition to rutile VO2 (VO2 (R)), which is accompanied by a metal-to-insulator transition. Bottom-up hydrothermal synthesis has proven to yield high quality monoclinic VO2 but requires toxic and highly reactive reducing agents that cannot be used outside of a research lab. Here, we present a new hydrothermal synthesis method using nontoxic and safe-to-use oxalic acid as a reducing agent for V2O5 to produce VO2 (M). In early stages of the process, polymorphs VO2 (A) and VO2 (B) were formed, which subsequently recrystallized to VO2 (M). Without the presence of W6+, this recrystallization did not occur. After a reaction time of 96 h at 230 °C in the presence of (NH4)6H2W12O40 in Teflon-lined rotated autoclaves, we realized highly crystalline, phase pure W-doped VO2 (M) microparticles of uniform size and asterisk shape (ΔH = 28.30 J·g-1, arm length = 6.7 ± 0.4 µm, arm width = 0.46 ± 0.06 µm). We extensively investigated the role of W6+ in the kinetics of formation of VO2 (M) and the thermodynamics of its structural phase transition.

Atg18 oligomer organization in assembled tubes and on lipid membrane scaffolds.

Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat. When reconstituted with lipid membranes, using subtomogram averaging we determined tilted Atg18 dimer structures bridging two juxtaposed lipid membranes spaced apart by 80 Å. Moreover, lipid reconstitution experiments further delineate the contributions of Atg18's FRRG motif and the amphipathic helical extension in membrane interaction. The observed structural plasticity of Atg18's oligomeric organization and membrane binding properties provide a molecular framework for the positioning of downstream components of the autophagy machinery.

ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.

Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome.

Carboxysomes are proteinaceous bacterial microcompartments that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating several enzymes, including ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural understanding of native carboxysomes is currently limited to low-resolution studies. Here, we report the characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryoelectron microscopy (cryo-EM). We have determined the structure of its RuBisCO enzyme, and obtained low-resolution maps of its icosahedral shell, and of its concentric interior organization. Using integrative modeling approaches, we have proposed a complete atomic model of an intact carboxysome, providing insight into its organization and assembly. This is critical for a better understanding of the carbon fixation mechanism and toward repurposing carboxysomes in synthetic biology for biotechnological applications.

Black-capped chickadees (Poecile atricapillus) discriminate between naturally-ordered and scramble-ordered chick-a-dee calls and individual preference is related to rate of learning.

Though many forms of animal communication are not reliant on the order in which components of signals are combined to be effective, there is evidence that order does matter for some communication systems. In the light of differential responding to calls of varying note-order observed in black-capped chickadees in the field, we set out to determine whether chickadees recognize syntactically-ordered and incorrectly-ordered chick-a-dee calls as separate and distinct conceptual categories using both an auditory preference task and go/no-go operant conditioning paradigm. Results show that chickadees spent more time on the perch that did not produce sound (i.e., silent perch) than on either of the acoustic perches (i.e., natural and scrambled order chick-a-dee call playback) and visited the perch associated with naturally-ordered calls more often than the perch associated with scrambled-order calls. Birds in both the True natural- and scrambled-order call groups continued to respond according to the contingencies that they learned in Discrimination training, indicating that black-capped chickadees are capable of perceiving and acting upon the categories of natural- versus scrambled-ordered calls.

Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution.

In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

Inadequate pitch-difference sensitivity prevents half of all listeners from discriminating major vs minor tone sequences.

Substantial evidence suggests that sensitivity to the difference between the major vs minor musical scales may be bimodally distributed. Much of this evidence comes from experiments using the "3-task." On each trial in the 3-task, the listener hears a rapid, random sequence of tones containing equal numbers of notes of either a G major or G minor triad and strives (with feedback) to judge which type of "tone-scramble" it was. This study asks whether the bimodal distribution in 3-task performance is due to variation (across listeners) in sensitivity to differences in pitch. On each trial in a "pitch-difference task," the listener hears two tones and judges whether the second tone is higher or lower than the first. When the first tone is roved (rather than fixed throughout the task), performance varies dramatically across listeners with median threshold approximately equal to a quarter-tone. Strikingly, nearly all listeners with thresholds higher than a quarter-tone performed near chance in the 3-task. Across listeners with thresholds below a quarter-tone, 3-task performance was uniformly distributed from chance to ceiling; thus, the large, lower mode of the distribution in 3-task performance is produced mainly by listeners with roved pitch-difference thresholds greater than a quarter-tone.

A polar bear paleogenome reveals extensive ancient gene flow from polar bears into brown bears.

Polar bears (Ursus maritimus) and brown bears (Ursus arctos) are sister species possessing distinct physiological and behavioural adaptations that evolved over the last 500,000 years. However, comparative and population genomics analyses have revealed that several extant and extinct brown bear populations have relatively recent polar bear ancestry, probably as the result of geographically localized instances of gene flow from polar bears into brown bears. Here, we generate and analyse an approximate 20X paleogenome from an approximately 100,000-year-old polar bear that reveals a massive prehistoric admixture event, which is evident in the genomes of all living brown bears. This ancient admixture event was not visible from genomic data derived from living polar bears. Like more recent events, this massive admixture event mainly involved unidirectional gene flow from polar bears into brown bears and occurred as climate changes caused overlap in the ranges of the two species. These findings highlight the complex reticulate paths that evolution can take within a regime of radically shifting climate.

Loss of left ventricular rotation is a significant determinant of functional mitral regurgitation.

AIM: To evaluate insufficient rotational movement of the left ventricle (LV) as a potential novel mechanism for functional regurgitation of the mitral valve (FMR). METHODS AND RESULTS: We compared reference subjects and patients with LV dysfunction (LVD, ejection fraction EF < 50%) with and without FMR (regurgitant volume RVol>10 ml). Subjects without structural mitral valve pathology undergoing cardiac MRI were evaluated. Delayed enhancement, global LV remodeling parameters, systolic twist and torsion were measured (using manual and novel automated cardiac MRI tissue-tracking). The study included 117 subjects with mean ± SD age 50.4 ± 17.8 years, of which 30.8% were female. Compared to subjects with LVD without FMR (n = 31), those with FMR (n = 37) had similar clinical characteristics, diagnoses, delayed enhancement, EF, and longitudinal strain. Subjects with FMR had significantly larger left ventricles (EDVi:136.6 ± 41.8 vs 97.5 ± 26.2 ml/m, p < 0.0001) with wider separation between papillary muscles (21.1 ± 7.6 vs 17.2 ± 5.7 mm, p = 0.023). Notably, they had lower apical (p < 0.0001) but not basal rotation and lower peak systolic twist (3.1 ± 2.4° vs 5.5 ± 2.5°, p < 0.0001) and torsion (0.56 ± 0.38°/cm vs 0.88 ± 0.52°/cm, p = 0.004). In a multivariate model for RVol including age, gender, twist, LV end-diastolic volume, sphericity index and separation between papillary muscles, only gender, volume and twist were significant. Twist was the most powerful correlate (beta -2.23, CI -3.26 to -1.23 p < 0.001). In patients with FMR, peak systolic twist negatively correlates with RVol (r = -0.73, p < 0.0001). CONCLUSION: Reduced rotational systolic LV motion is significantly and independently associated with RVol among patients with FMR, suggesting a novel pathophysiological mechanism and a potential therapeutic target.

The structure of the bacterial DNA segregation ATPase filament reveals the conformational plasticity of ParA upon DNA binding.

The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA's cooperative binding to DNA and the formation of high ParA density regions on the nucleoid.

Lifetime mobility of an Arctic woolly mammoth.

Little is known about woolly mammoth (Mammuthus primigenius) mobility and range. Here we use high temporal resolution sequential analyses of strontium isotope ratios along an entire 1.7-meter-long tusk to reconstruct the movements of an Arctic woolly mammoth that lived 17,100 years ago, during the last ice age. We use an isotope-guided random walk approach to compare the tusk's strontium and oxygen isotope profiles to isotopic maps. Our modeling reveals patterns of movement across a geographically extensive range during the animal's ~28-year life span that varied with life stages. Maintenance of this level of mobility by megafaunal species such as mammoth would have been increasingly difficult as the ice age ended and the environment changed at high latitudes.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii.

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

Structural interpretation of cryo-EM image reconstructions.

The productivity of single-particle cryo-EM as a structure determination method has rapidly increased as many novel biological structures are being elucidated. The ultimate result of the cryo-EM experiment is an atomic model that should faithfully represent the computed image reconstruction. Although the principal approach of atomic model building and refinement from maps resembles that of the X-ray crystallographic methods, there are important differences due to the unique properties resulting from the 3D image reconstructions. In this review, we discuss the practiced work-flow from the cryo-EM image reconstruction to the atomic model. We give an overview of (i) resolution determination methods in cryo-EM including local and directional resolution variation, (ii) cryo-EM map contrast optimization including complementary map types that can help in identifying ambiguous density features, (iii) atomic model building and (iv) refinement in various resolution regimes including (v) their validation and (vi) discuss differences between X-ray and cryo-EM maps. Based on the methods originally developed for X-ray crystallography, the path from 3D image reconstruction to atomic coordinates has become an integral and important part of the cryo-EM structure determination work-flow that routinely delivers atomic models.

Oligoglycidol-Functionalised Styrene Macromolecules as Reactive Surfactants in the Emulsion Polymerisation of Styrene: The Impact of Chain Length and Concentration on Particle Size and Colloidal Stability.

Reactive surfactants (surfmers), which are covalently attached to the surface of sub-micron sized polymer particles during emulsion polymerisation, are applied to tailor the surface functionality of polymer particles for an application of choice. We present a systematic study on the use of oligoglycidol-functionalised styrene macromolecules as surfmers in the emulsion polymerization of styrene. Firstly, we report the impact of the surfmer concentration on the particle size for polymerisations performed above and below the critical micelle concentration. Secondly, we report the influence of the oligoglycidol chain length on the particle size. Thirdly, we conducted experiments to analyse the influence of the surfmer concentration and its chain length on the colloidal stability of the aqueous polystyrene nanoparticles in sodium chloride solutions. We demonstrated that the size of polystyrene particles could be influenced by changing both the surfmer concentration and its chain length. Furthermore, we showed that the colloidal stability of the oligoglycidol-functionalized polystyrene particles is dependent on the particle size, and not directly related to the oligoglycidol chain length.

A model of the uncertainty effects in choice reaction time that includes a major contribution from effector selection.

Hick's law describes the relation between choice reaction time (RT) and the number of stimulus-response alternatives (NA). For over half a century, this uncertainty effect has been ascribed primarily to the time taken to map a stimulus to its associated response. Here, data from 2 experiments suggests that selection of the appropriate effector-the particular body part to make a response-also contributes substantially to the uncertainty effect. This insight is important both for our understanding of basic cognitive architecture and because many classic experiments studying stimulus-response mapping have confounded NA with the number of effectors. Our data also suggest that, when stimuli are spatial and linked to the responses in an intuitively simple layout, the time required for stimulus-response mapping depends minimally on the NA, independent of effector. Experiment 1 demonstrated that in order to account for the complex patterns of uncertainty effects observed when stimulus type (spatial vs. symbolic), response mode (typing, with multiple effectors vs. touching with a single, known effector), and participant population (skilled vs. novice typists) are all manipulated a model is required that includes effector selection, along with stimulus-response mapping, and a proper treatment of stimulus-response repetitions. Using spatial indicator stimuli that minimized the contributions of stimulus-response mapping, Experiment 2 compared 4 effector conditions-the factorial combination of 1 or 3 fingers on one or both hands. The results showed that the increase in the uncertainty effect associated with the number of effectors is negatively accelerated and possibly additive across the variation of hands and fingers. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Climate-driven ecological stability as a globally shared cause of Late Quaternary megafaunal extinctions: the Plaids and Stripes Hypothesis.

Controversy persists about why so many large-bodied mammal species went extinct around the end of the last ice age. Resolving this is important for understanding extinction processes in general, for assessing the ecological roles of humans, and for conserving remaining megafaunal species, many of which are endangered today. Here we explore an integrative hypothesis that asserts that an underlying cause of Late Quaternary megafaunal extinctions was a fundamental shift in the spatio-temporal fabric of ecosystems worldwide. This shift was triggered by the loss of the millennial-scale climate fluctuations that were characteristic of the ice age but ceased approximately 11700 years ago on most continents. Under ice-age conditions, which prevailed for much of the preceding 2.6 Ma, these radical and rapid climate changes prevented many ecosystems from fully equilibrating with their contemporary climates. Instead of today's 'striped' world in which species' ranges have equilibrated with gradients of temperature, moisture, and seasonality, the ice-age world was a disequilibrial 'plaid' in which species' ranges shifted rapidly and repeatedly over time and space, rarely catching up with contemporary climate. In the transient ecosystems that resulted, certain physiological, anatomical, and ecological attributes shared by megafaunal species pre-adapted them for success. These traits included greater metabolic and locomotory efficiency, increased resistance to starvation, longer life spans, greater sensory ranges, and the ability to be nomadic or migratory. When the plaid world of the ice age ended, many of the advantages of being large were either lost or became disadvantages. For instance in a striped world, the low population densities and slow reproductive rates associated with large body size reduced the resiliency of megafaunal species to population bottlenecks. As the ice age ended, the downsides of being large in striped environments lowered the extinction thresholds of megafauna worldwide, which then increased the vulnerability of individual species to a variety of proximate threats they had previously tolerated, such as human predation, competition with other species, and habitat loss. For many megafaunal species, the plaid-to-stripes transition may have been near the base of a hierarchy of extinction causes whose relative importances varied geographically, temporally, and taxonomically.