Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
Increasing empirical evidence supports associations between neighborhood environments and physical activity. However, since most studies were conducted in a single country, particularly western countries, the generalizability of associations in an international setting is not well understood. The current study examined whether associations between perceived attributes of neighborhood environments and physical activity differed by country.
Methods
Population representative samples from 11 countries on five continents were surveyed using comparable methodologies and measurement instruments. Neighborhood environment × country interactions were tested in logistic regression models with meeting physical activity recommendations as the outcome, adjusted for demographic characteristics. Country-specific associations were reported.
Results
Significant neighborhood environment attribute × country interactions implied some differences across countries in the association of each neighborhood attribute with meeting physical activity recommendations. Across the 11 countries, land-use mix and sidewalks had the most consistent associations with physical activity. Access to public transit, bicycle facilities, and low-cost recreation facilities had some associations with physical activity, but with less consistency across countries. There was little evidence supporting the associations of residential density and crime-related safety with physical activity in most countries.
Conclusion
There is evidence of generalizability for the associations of land use mix, and presence of sidewalks with physical activity. Associations of other neighborhood characteristics with physical activity tended to differ by country. Future studies should include objective measures of neighborhood environments, compare psychometric properties of reports across countries, and use better specified models to further understand the similarities and differences in associations across countries.
Neighborhood environment studies of physical activity (PA) have been mainly single-country focused. The International Prevalence Study (IPS) presented a rare opportunity to examine neighborhood features across countries. The purpose of this analysis was to: 1) detect international neighborhood typologies based on participants’ response patterns to an environment survey and 2) to estimate associations between neighborhood environment patterns and PA.
Methods
A Latent Class Analysis (LCA) was conducted on pooled IPS adults (N=11,541) aged 18 to 64 years old (mean=37.5 ±12.8 yrs; 55.6% women) from 11 countries including Belgium, Brazil, Canada, Colombia, Hong Kong, Japan, Lithuania, New Zealand, Norway, Sweden, and the U.S. This subset used the Physical Activity Neighborhood Environment Survey (PANES) that briefly assessed 7 attributes within 10–15 minutes walk of participants’ residences, including residential density, access to shops/services, recreational facilities, public transit facilities, presence of sidewalks and bike paths, and personal safety. LCA derived meaningful subgroups from participants’ response patterns to PANES items, and participants were assigned to neighborhood types. The validated short-form International Physical Activity Questionnaire (IPAQ) measured likelihood of meeting the 150 minutes/week PA guideline. To validate derived classes, meeting the guideline either by walking or total PA was regressed on neighborhood types using a weighted generalized linear regression model, adjusting for gender, age and country.
Results
A 5-subgroup solution fitted the dataset and was interpretable. Neighborhood types were labeled, “Overall Activity Supportive (52% of sample)”, “High Walkable and Unsafe with Few Recreation Facilities (16%)”, “Safe with Active Transport Facilities (12%)”, “Transit and Shops Dense with Few Amenities (15%)”, and “Safe but Activity Unsupportive (5%)”. Country representation differed by type (e.g., U.S. disproportionally represented “Safe but Activity Unsupportive”). Compared to the Safe but Activity Unsupportive, two types showed greater odds of meeting PA guideline for walking outcome (High Walkable and Unsafe with Few Recreation Facilities, OR= 2.26 (95% CI 1.18-4.31); Overall Activity Supportive, OR= 1.90 (95% CI 1.13-3.21). Significant but smaller odds ratios were also found for total PA.
Conclusions
Meaningful neighborhood patterns generalized across countries and explained practical differences in PA. These observational results support WHO/UN recommendations for programs and policies targeted to improve features of the neighborhood environment for PA.
The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.
Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.
This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of ∼40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from ∼35 to ∼300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 × 1012 photons per µm2 per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
