Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Dynamics of rare gas solids irradiated by electron beams.

The remarkable success of x-ray free-electron lasers and their ability to image biological macromolecules while outrunning secondary radiation damage due to photoelectrons, by using femtosecond pulses, raise the question of whether this can be done using pulsed high-energy electron beams. In this paper, we use excited state molecular dynamics simulations, with tabulated potentials, for rare gas solids to investigate the effect of radiation damage due to inelastic scattering (by plasmons, excitons, and heat) on the pair distribution function. We use electron energy loss spectra to characterize the electronic excitations responsible for radiation damage.

The short-term effects of a mass reach physical activity campaign: an evaluation using hierarchy of effects model and intention profiles.

BACKGROUND: Mass reach physical activity campaigns are designed to deliver physical-activity related messages to a large population across different media including print, television, radio, and websites. Few evaluations have examined the short-term effects of a mass reach campaign on participants who were engaged with the campaign. The current research examined the short-term effects of the ParticipACTION 150 Play List, a mass reach physical activity campaign, on participants who registered with the campaign website. METHODS: Participants (N = 7801) completed a registration questionnaire measuring demographic information, awareness and recall of physical activity and sport advertising, and self-reported number of activities tried or planned to try from the 150 Play List. A follow-up survey was completed by 1298 participants from the original sample. Additional questions assessed experience with the 150 Play List and attitudes towards campaign advertisements. RESULTS: Approximately 14.5% of participants cited the ParticipACTION 150 Play List and 23.6% mentioned a 'getting active' message when recalling advertisements. Those who named the 150 Play List or getting active reported more activities tried and more activities planned than those who did not. They were also more likely to say they had tried a new activity and planned ongoing participation. It was also found that participants with a disability were more likely to have tried a new activity compared to those not in a minority group. Other correlates of trying new activities at follow-up were younger age, more positive reported experience with the 150 Play List, and more favourable attitudes towards campaign advertisements. Those who did not intend continued participation, or who were unsure at baseline and then decided against continued participation at follow-up, reported they were less sedentary or encouraging others to be active. CONCLUSIONS: This research addresses the gap in evidence regarding the efficacy of mass reach physical activity campaigns by informing whether a year-long campaign like the 150 Play List can be effective in influencing the behavior of those engaged with the campaign. The results reinforce the idea that 'top of mind' awareness should be measured. Investigating intention profiles can help inform campaign impacts and continuation intentions.

Short-wavelength free-electron laser sources and science: a review.

This review is focused on free-electron lasers (FELs) in the hard to soft x-ray regime. The aim is to provide newcomers to the area with insights into: the basic physics of FELs, the qualities of the radiation they produce, the challenges of transmitting that radiation to end users and the diversity of current scientific applications. Initial consideration is given to FEL theory in order to provide the foundation for discussion of FEL output properties and the technical challenges of short-wavelength FELs. This is followed by an overview of existing x-ray FEL facilities, future facilities and FEL frontiers. To provide a context for information in the above sections, a detailed comparison of the photon pulse characteristics of FEL sources with those of other sources of high brightness x-rays is made. A brief summary of FEL beamline design and photon diagnostics then precedes an overview of FEL scientific applications. Recent highlights are covered in sections on structural biology, atomic and molecular physics, photochemistry, non-linear spectroscopy, shock physics, solid density plasmas. A short industrial perspective is also included to emphasise potential in this area.

The extraction of single-particle diffraction patterns from a multiple-particle diffraction pattern.

The structures of biological molecules may soon be determined with X-ray free-electron lasers without crystallization by recording the coherent diffraction patterns of many identical copies of a molecule. Most analysis methods require a measurement of each molecule individually. However, current injection methods deliver particles to the X-ray beam stochastically and the maximum yield of single particle measurements is 37% at optimal concentration. The remaining 63% of pulses intercept no particles or multiple particles. We demonstrate that in the latter case single particle diffraction patterns can be extracted provided the particles are sufficiently separated. The technique has the potential to greatly increase the amount of data available for three-dimensional imaging of identical particles with X-ray lasers.

X-ray lasers for structural and dynamic biology.

Research opportunities and techniques are reviewed for the application of hard x-ray pulsed free-electron lasers (XFEL) to structural biology. These include the imaging of protein nanocrystals, single particles such as viruses, pump--probe experiments for time-resolved nanocrystallography, and snapshot wide-angle x-ray scattering (WAXS) from molecules in solution. The use of femtosecond exposure times, rather than freezing of samples, as a means of minimizing radiation damage is shown to open up new opportunities for the molecular imaging of biochemical reactions at room temperature in solution. This is possible using a 'diffract-and-destroy' mode in which the incident pulse terminates before radiation damage begins. Methods for delivering hundreds of hydrated bioparticles per second (in random orientations) to a pulsed x-ray beam are described. New data analysis approaches are outlined for the correlated fluctuations in fast WAXS, for protein nanocrystals just a few molecules on a side, and for the continuous x-ray scattering from a single virus. Methods for determining the orientation of a molecule from its diffraction pattern are reviewed. Methods for the preparation of protein nanocrystals are also reviewed. New opportunities for solving the phase problem for XFEL data are outlined. A summary of the latest results is given, which now extend to atomic resolution for nanocrystals. Possibilities for time-resolved chemistry using fast WAXS (solution scattering) from mixtures is reviewed, toward the general goal of making molecular movies of biochemical processes.

X-ray diffraction from membrane protein nanocrystals.

Membrane proteins constitute > 30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is < 300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 µm. The results demonstrate that there are membrane protein crystals that contain < 100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain < 200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.

New light on disordered ensembles: ab initio structure determination of one particle from scattering fluctuations of many copies.

We report on the first experimental ab initio reconstruction of an image of a single particle from fluctuations in the scattering from an ensemble of copies, randomly oriented about an axis. The method is applicable to identical particles frozen in space or time (as by snapshot diffraction from an x-ray free electron laser). These fluctuations enhance information obtainable from an experiment such as conventional small angle x-ray scattering.

A new solution to the curved Ewald sphere problem for 3D image reconstruction in electron microscopy.

We develop an algorithm capable of imaging a three-dimensional object given a collection of two-dimensional images of that object that are significantly influenced by the curvature of the Ewald sphere. These two-dimensional images cannot be approximated as projections of the object. Such an algorithm is useful in cryo-electron microscopy where larger samples, higher resolution, or lower energy electron beams are desired, all of which contribute to the significance of Ewald curvature.

Quantitative agreement between dynamical rocking curves in ultrafast electron diffraction for x-ray lasers.

Electron diffraction through a thin patterned silicon membrane can be used to create complex spatial modulations in electron distributions. By precisely varying parameters such as crystallographic orientation and wafer thickness, the intensity of reflections in the diffraction plane can be controlled and by placing an aperture to block all but one spot, we can form an image with different parts of the patterned membrane, as is done for bright-field imaging in microscopy. The patterned electron beams can then be used to control phase and amplitude of subsequent x-ray emission, enabling novel coherent x-ray methods. The electrons themselves can also be used for femtosecond time resolved diffraction and microscopy. As a first step toward patterned beams, we demonstrate experimentally and through simulation the ability to accurately predict and control diffraction spot intensities. We simulate MeV transmission electron diffraction patterns using the multislice method for various crystallographic orientations of a single crystal Si(001) membrane near beam normal. The resulting intensity maps of the Bragg reflections are compared to experimental results obtained at the Accelerator Structure Test Area Ultrafast Electron Diffraction (ASTA UED) facility at SLAC. Furthermore, the fraction of inelastic and elastic scattering of the initial charge is estimated along with the absorption of the membrane to determine the contrast that would be seen in a patterned version of the Si(001) membrane.

Changes in BMI over 6 years: the role of demographic and neighborhood characteristics.

OBJECTIVE: To undertake a 6-year longitudinal investigation of the relationship between the built environment (perceived and objectively measured) and change in body mass index (BMI). Specifically, this research examined whether change in BMI was predicted by objectively measured neighborhood walkability and socioeconomic status (SES), and perceived neighborhood characteristics (for example, crime, traffic and interesting things to look at) in addition to other factors such as age, gender, education, physical activity, fruit and vegetable consumption and smoking. DESIGN: Longitudinal study. SUBJECTS: 500 adults who provided complete data in 2002 and 2008 and who did not move over the course of the study (47.8% female; age in 2002: 18-90 years). MEASUREMENTS: Telephone surveys in 2002 and 2008 measuring perceptions of their neighborhood environment and demographic factors. Objective measures of neighborhood characteristics were calculated using census data and geographical information systems in 2006. RESULTS: Age, neighborhood SES and perceived traffic were significantly related to increased BMI over the 6 years. Younger participants and those in lower SES neighborhoods were more likely to have increased BMI. Agreement with the statement that traffic made it difficult to walk also predicted increased BMI. CONCLUSION: This study adds to the literature to show that BMI increased in low SES neighborhoods. Although more research is needed to fully understand how neighborhood SES contributes to obesity, it is without question that individuals in socially disadvantaged neighborhoods face more barriers to health than their wealthier counterparts. This study also calls into question the relationship between walkability and changes in BMI and emphasizes the necessity of longitudinal data rather than relying on cross-sectional research.

Crystal site location of iron and trace elements in a magnesium-iron olivine by a new crystallographic technique.

A new crysallographic technique has been developed, which has been applied to the problem of locating the cations in a natural olivine crystal with the composition (Mg(0.90)Fe(0.10)Ni(0.004)Mn(0.002))(2)SiO(4). The method uses the variation of characteristic x-ray emission with the direction of an exciting electron beam in an analytical transmission electron microscope. It may be applied to nanometer-sized areas and to concentrations as low as 0.1 atomic percent, is capable of distinguishing neighbors in the periodic table, and does not require external standards. The iron atoms in this crystal are evenly distributed between the two available crystal sites M1 and M2 (49.6 +/- 1 percent on M1), whereas the trace elements nickel and manganese occupy the M1 and M2 positions, respectively (97 +/- 5 percent nickel on M1 and 1 +/- 5 percent manganese on M1).

An assessment of the resolution limitation due to radiation-damage in x-ray diffraction microscopy.

X-ray diffraction microscopy (XDM) is a new form of x-ray imaging that is being practiced at several third-generation synchrotron-radiation x-ray facilities. Nine years have elapsed since the technique was first introduced and it has made rapid progress in demonstrating high-resolution three-dimensional imaging and promises few-nm resolution with much larger samples than can be imaged in the transmission electron microscope. Both life- and materials-science applications of XDM are intended, and it is expected that the principal limitation to resolution will be radiation damage for life science and the coherent power of available x-ray sources for material science. In this paper we address the question of the role of radiation damage. We use a statistical analysis based on the so-called "dose fractionation theorem" of Hegerl and Hoppe to calculate the dose needed to make an image of a single life-science sample by XDM with a given resolution. We find that for simply-shaped objects the needed dose scales with the inverse fourth power of the resolution and present experimental evidence to support this finding. To determine the maximum tolerable dose we have assembled a number of data taken from the literature plus some measurements of our own which cover ranges of resolution that are not well covered otherwise. The conclusion of this study is that, based on the natural contrast between protein and water and "Rose-criterion" image quality, one should be able to image a frozen-hydrated biological sample using XDM at a resolution of about 10 nm.

Neighborhood and developmental differences in children's perceptions of opportunities for play and physical activity.

The purposes of this study were to examine perceptions of places to play and be physically active among children from two different urban neighborhoods, and evaluate these perceptions for age-related developmental differences. One hundred and sixty-eight children from grades K-6 (aged 6-12 years old) completed mental maps depicting places where they could play and be physically active. The children were recruited from schools in two neighborhoods-one a high-walkability (H-W) grid-style neighborhood, the other a low-walkability (L-W) lollipop-style (i.e., cul-de-sacs) neighborhood. Analysis revealed that children in the H-W neighborhood depicted more active transportation and less non-active transportation than children in the L-W neighborhood. Children in the lowest grades (K-2) in the L-W neighborhood depicted more play in the home/yard environment than the oldest children, more good weather image events than children in Grades 3-6, and less play outside the home/yard environment than children in Grades 3 and 4. In the H-W neighborhood, the youngest children (K-2) depicted significantly less play in the home/yard environment and less play outside the home/yard environment than older children (Grades 3-6). Thus, both the type of urban neighborhood and children's age moderated perceptions of places to play and be physically active.

XFELs for structure and dynamics in biology.

The development and application of the free-electron X-ray laser (XFEL) to structure and dynamics in biology since its inception in 2009 are reviewed. The research opportunities which result from the ability to outrun most radiation-damage effects are outlined, and some grand challenges are suggested. By avoiding the need to cool samples to minimize damage, the XFEL has permitted atomic resolution imaging of molecular processes on the 100 fs timescale under near-physiological conditions and in the correct thermal bath in which molecular machines operate. Radiation damage, comparisons of XFEL and synchrotron work, single-particle diffraction, fast solution scattering, pump-probe studies on photosensitive proteins, mix-and-inject experiments, caged molecules, pH jump and other reaction-initiation methods, and the study of molecular machines are all discussed. Sample-delivery methods and data-analysis algorithms for the various modes, from serial femtosecond crystallo-graphy to fast solution scattering, fluctuation X-ray scattering, mixing jet experiments and single-particle diffraction, are also reviewed.

How perceptions of community environment influence health behaviours: using the Analysis Grid for Environments Linked to Obesity Framework as a mechanism for exploration. / Perceptions à l'égard de l'environnement et comportements en matière de santé : étude fondée sur le cadre ANGELO (grille d'analyse des environnements liés à l'obésité).

INTRODUCTION: Overweight and obesity are influenced by a complex interplay of individual and environmental factors that affect physical activity and healthy eating. Nevertheless, little has been reported on people's perceptions of those factors. Addressing this critical gap and community partner needs, this study explored how people perceived the influence of micro- and macroenvironmental factors on physical activity and healthy eating. METHODS: Community partners wanted the study results in a format that would be readily and easily used by local decision makers. We used photovoice to engage 35 community members across four municipalities in Alberta, Canada, and to share their narratives about their physical activity and healthy eating. A combination of inductive and deductive analysis categorized data by environmental level (micro vs. macro) and type (physical, political, economic, and sociocultural), guided by the Analysis Grid for Environments Linked to Obesity Framework. RESULTS: Participants conceptualized health-influencing factors more broadly than physical activity and healthy eating to include "community social health." Participants spoke most often about the influence of the microenvironment (n = 792 ANGELO Framework coding tallies) on their physical activity, healthy eating and community social health in comparison to the macroenvironment (n = 93). Photovoice results provided a visual narrative to community partners and decision makers about how people's ability to make healthy choices can be limited by macroenvironmental forces beyond their control. CONCLUSION: Focussing future research on macro- and microenvironmental influences and localized community social health can inform practice by providing strategies on how to implement healthy changes within communities, while ensuring that research and interventions echo diverse people's perceptions.

INTRODUCTION: Le surpoids et l'obésité dépendent de l'interaction complexe entre facteurs liés à l'indivu et facteurs liés à l'environnement ayant une influence sur l'activité physique et une alimentation saine. Il existe pourtant peu d'information sur les perceptions des individus à l'égard de ces facteurs. Afin de combler cette lacune importante et de répondre aux besoins de nos partenaires des collectivités, nous examinons comment les individus perçoivent l'influence des facteurs micro- et macroenvironnementaux sur leur activité physique et leur alimentation. MÉTHODOLOGIE: Nos partenaires des collectivités souhaitaient que les résultats de l'étude soient diffusés dans un format facile à utiliser par les décideurs locaux. Avec la méthode photovoix, nous avons motivé 35 membres issus de quatre collectivités de l'Alberta (Canada) à fournir leurs témoignages à propos de leur activité physique et d'une alimentation saine. Nous avons employé une combinaison d'analyses inductives et déductives pour classer les données par niveau (micro et macro) d'environnement et par type (environnement physique, politique, économique et socioculturel), à l'aide de la Grille d'analyse des environnements liés à l'obésité (ANGELO). RÉSULTATS: Pour les participants, les facteurs liés à la santé étaient plus larges que l'activité physique et une alimentation saine, incluant la « santé sociocommunautaire ¼. Les participants ont parlé plus souvent de l'influence du microenvironnement (score de 792 après codage au moyen du cadre ANGELO) sur l'activité physique, une alimentation saine ou la santé sociocommunautaire que sur l'influence du macroenvironnement (score de 93). Les résultats obtenus avec la méthode photovoix ont fourni aux partenaires des collectivités et aux décideurs un témoignage visuel de la manière dont la capacité des individus à faire des choix sains peut être restreinte par des forces macroenvironnementales qui échappent à leur contrôle. CONCLUSION: Axer les recherches ultérieures sur les influences et macroenvironnementales et microenvironnementales ainsi que sur la santé sociocommunautaire locale sera utile, car cela alimentera les stratégies de changement en vue d'améliorer la santé des collectivités tout en garantissant que la recherche et les interventions font écho aux perceptions diverses de la population.

Reconstruction of complex single-particle images using charge-flipping algorithm.

An iterative algorithm is developed to retrieve the complex exit-face wavefunction for a two-dimensional projection of a nanoparticle from a measurement of the oversampled modulus of its Fourier transform in reciprocal space. The algorithm does not require the support (boundary) of the object to be known. A loose support for the complex object is gradually found using the Oszlanyi-Suto charge-flipping algorithm, and a compact support is then iteratively developed using a dynamic Gerchberg-Saxton-Fienup algorithm. At the same time, the complex object is reconstructed using this compact support. The algorithm applies to the reconstruction of complex images with any distribution of phase values from 0 to 2pi. Modification of the algorithm by using real-value constraints for a complex object in the charge-flipping algorithm leads to faster reconstruction of the object whose phase value is smaller than pi/2.

Diffraction and imaging from a beam of laser-aligned proteins: resolution limits.

The effect of the limited alignment of hydrated molecules is considered in a laser-aligned molecular beam, on diffraction patterns taken from the beam. Simulated patterns for a protein beam are inverted using the Fienup-Gerchberg-Saxton phasing algorithm, and the effect of limited alignment on the resolution of the resulting potential maps is studied. For a typical protein molecule (lysozyme) with anisotropic polarizability, it is found that up to 1 kW of continuous-wave near-infrared laser power (depending on dielectric constant), together with cooling to liquid-nitrogen temperatures, may be needed to produce sufficiently accurate alignment for direct observation of the secondary structure of proteins in the reconstructed potential or charge-density map. For a typical virus (TMV), a 50 W continuous-wave laser is adequate for subnanometre resolution at room temperature. The dependence of resolution on laser power, temperature, molecular size, shape and dielectric constant is analyzed.

Exploring women's responses to online media coverage of weight loss surgery.

The purpose of this study was to understand the reactions of women to online news articles about weight loss surgery and related reader comments. Focus groups were conducted; open-ended questions were asked to elicit responses to existing online news media content related to weight loss surgery. The participants described the online articles as predominantly supportive of weight loss surgery and in response they expressed a desire to see more critical content, including different and competing perspectives. Participants felt the online comments represented extreme perspectives and were predominately negative. These were therefore not viewed as helpful or informative. Nevertheless, readers viewed comments as a form of entertainment. Because of the aggressive and anonymous nature of reader comments in response to online news stories, the participants did not feel comfortable leaving comments themselves on the news sites. Findings highlight the importance of gathering readers' perspectives in response to interactive media content and, in particular, health information.

The linac coherent light source single particle imaging road map.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.