Alignment of cryo-EM movies of individual particles by optimization of image translations

Direct detector device (DDD) cameras have revolutionized single particle electron cryomicroscopy (cryo-EM). In addition to an improved camera detective quantum efficiency, acquisition of DDD movies allows for correction of movement of the specimen, due both to instabilities in the microscope specimen stage and electron beam-induced movement. Unlike specimen stage drift, beam-induced movement is not always homogeneous within an image. Local correlation in the trajectories of nearby particles suggests that beam-induced motion is due to deformation of the ice layer. Algorithms have already been described that can correct movement for large regions of frames and for > 1 MDa protein particles. Another algorithm allows individual < 1 MDa protein particle trajectories to be estimated, but requires rolling averages to be calculated from frames and fits linear trajectories for particles. Here we describe an algorithm that allows for individual < 1 MDa particle images to be aligned without frame averaging or linear trajectories. The algorithm maximizes the overall correlation of the shifted frames with the sum of the shifted frames. The optimum in this single objective function is found efficiently by making use of analytically calculated derivatives of the function. To smooth estimates of particle trajectories, rapid changes in particle positions between frames are penalized in the objective function and weighted averaging of nearby trajectories ensures local correlation in trajectories. This individual particle motion correction, in combination with weighting of Fourier components to account for increasing radiation damage in later frames, can be used to improve 3-D maps from single particle cryo-EM.Comment: 11 pages, 4 figure

Validating maps from single particle electron cryomicroscopy

Progress in single particle cryo-EM, most recently due to the introduction of direct detector devices, has made the high-resolution structure determination of biological assemblies smaller than 500kDa more routine, but has also increased attention on the need for tools to demonstrate the validity of single particle maps. Although map validation is a continuing subject of research, some consensus has been reached on procedures that reduce model bias and over-fitting during map refinement as well as specific tests that demonstrate map validity. Tilt-pair analysis may be used as a method for demonstrating the consistency at low resolution of a map with image data. For higher-resolution maps, new procedures for more robust resolution assessment and for validating the refinement of atomic coordinate models into single particle maps have been developed

Skating on a Film of Air: Drops Impacting on a Surface

Drops impacting on a surface are ubiquitous in our everyday experience. This impact is understood within a commonly accepted hydrodynamic picture: it is initiated by a rapid shock and a subsequent ejection of a sheet leading to beautiful splashing patterns. However, this picture ignores the essential role of the air that is trapped between the impacting drop and the surface. Here we describe a new imaging modality that is sensitive to the behavior right at the surface. We show that a very thin film of air, only a few tens of nanometers thick, remains trapped between the falling drop and the surface as the drop spreads. The thin film of air serves to lubricate the drop enabling the fluid to skate on the air film laterally outward at surprisingly high velocities, consistent with theoretical predictions. Eventually this thin film of air must break down as the fluid wets the surface. We suggest that this occurs in a spinodal-like fashion, and causes a very rapid spreading of a wetting front outwards; simultaneously the wetting fluid spreads inward much more slowly, trapping a bubble of air within the drop. Our results show that the dynamics of impacting drops are much more complex than previously thought and exhibit a rich array of unexpected phenomena that require rethinking classical paradigms.Comment: 4 pages, 4 figure

Probing the Inner Disk Emission of the Herbig Ae Stars HD 163296 and HD 190073

The physical processes occurring within the inner few astronomical units of proto-planetary disks surrounding Herbig Ae stars are crucial to setting the environment in which the outer planet-forming disk evolves and put critical constraints on the processes of accretion and planet migration. We present the most complete published sample of high angular resolution H- and K-band observations of the stars HD 163296 and HD 190073, including 30 previously unpublished nights of observations of the former and 45 nights of the latter with the CHARA long-baseline interferometer, in addition to archival VLTI data. We confirm previous observations suggesting significant near-infrared emission originates within the putative dust evaporation front of HD 163296 and show this is the case for HD 190073 as well. The H- and K-band sizes are the same within $(3 \pm 3)\%$ for HD 163296 and within $(6 \pm 10)\%$ for HD 190073. The radial surface brightness profiles for both disks are remarkably Gaussian-like with little or no sign of the sharp edge expected for a dust evaporation front. Coupled with spectral energy distribution analysis, our direct measurements of the stellar flux component at H and K bands suggest that HD 190073 is much younger (<400 kyr) and more massive (~5.6 M$_\odot$) than previously thought, mainly as a consequence of the new Gaia distance (891 pc).Comment: 19 pages, 6 figure

Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents

Optically active nanomaterials promise to advance a range of biophotonic techniques through nanoscale optical effects and integration of multiple imaging and therapeutic modalities. Here, we report the development of porphysomes; nanovesicles formed from self-assembled porphyrin bilayers that generated large, tunable extinction coefficients, structure-dependent fluorescence self-quenching and unique photothermal and photoacoustic properties. Porphysomes enabled the sensitive visualization of lymphatic systems using photoacoustic tomography. Near-infrared fluorescence generation could be restored on dissociation, creating opportunities for low-background fluorescence imaging. As a result of their organic nature, porphysomes were enzymatically biodegradable and induced minimal acute toxicity in mice with intravenous doses of 1,000 mg kg^(−1). In a similar manner to liposomes, the large aqueous core of porphysomes could be passively or actively loaded. Following systemic administration, porphysomes accumulated in tumours of xenograft-bearing mice and laser irradiation induced photothermal tumour ablation. The optical properties and biocompatibility of porphysomes demonstrate the multimodal potential of organic nanoparticles for biophotonic imaging and therapy

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases

Sex difference in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic POMC neurons.

OBJECTIVE: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified. METHODS: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT 2C R (CRE) mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable Pomc (NEO) mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight. RESULTS: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity. CONCLUSIONS: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual