Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA)

Imaging single cells in a beam of live cyanobacteria with an X-ray laser

Citation: van der Schot, G., Svenda, M., Maia, F., Hantke, M., DePonte, D. P., Seibert, M. M., . . . Ekeberg, T. (2015). Imaging single cells in a beam of live cyanobacteria with an X-ray laser. Nature Communications, 6, 9. doi:10.1038/ncomms6704There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.Additional Authors: Almeida, N. F.;Odic, D.;Hasse, D.;Carlsson, G. H.;Larsson, D. S. D.;Barty, A.;Martin, A. V.;Schorb, S.;Bostedt, C.;Bozek, J. D.;Rolles, D.;Rudenko, A.;Epp, S.;Foucar, L.;Rudek, B.;Hartmann, R.;Kimmel, N.;Holl, P.;Englert, L.;Loh, N. T. D.;Chapman, H. N.;Andersson, I.;Hajdu, J.;Ekeberg, T

Performance of the WeNMR CS-Rosetta3 web server in CASD-NMR

We present here the performance of the WeNMR CS-Rosetta3 web server in CASD-NMR, the critical assessment of automated structure determination by NMR. The CS-Rosetta server uses only chemical shifts for structure prediction, in combination, when available, with a post-scoring procedure based on unassigned NOE lists (Huang et al. in J Am Chem Soc 127:1665-1674, 2005b, doi: 10.1021/ja047109h). We compare the original submissions using a previous version of the server based on Rosetta version 2.6 with recalculated targets using the new R3FP fragment picker for fragment selection and implementing a new annotation of prediction reliability (van der Schot et al. in J Biomol NMR 57:27-35, 2013, doi: 10.1007/s10858-013-9762-6), both implemented in the CS-Rosetta3 WeNMR server. In this second round of CASD-NMR, the WeNMR CS-Rosetta server has demonstrated a much better performance than in the first round since only converged targets were submitted. Further, recalculation of all CASD-NMR targets using the new version of the server demonstrates that our new annotation of prediction quality is giving reliable results. Predictions annotated as weak are often found to provide useful models, but only for a fraction of the sequence, and should therefore only be used with caution

Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser

Citation: Ekeberg, T., Svenda, M., Abergel, C., Maia, F., Seltzer, V., Claverie, J. M., . . . Hajdu, J. (2015). Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser. Physical Review Letters, 114(9), 6. doi:10.1103/PhysRevLett.114.098102We present a proof-of-concept three-dimensional reconstruction of the giant mimivirus particle from experimentally measured diffraction patterns from an x-ray free-electron laser. Three-dimensional imaging requires the assembly of many two-dimensional patterns into an internally consistent Fourier volume. Since each particle is randomly oriented when exposed to the x-ray pulse, relative orientations have to be retrieved from the diffraction data alone. We achieve this with a modified version of the expand, maximize and compress algorithm and validate our result using new methods.Additional Authors: Andersson, I.;Loh, N. D.;Martin, A. V.;Chapman, H.;Bostedt, C.;Bozek, J. D.;Ferguson, K. R.;Krzywinski, J.;Epp, S. W.;Rolles, D.;Rudenko, A.;Hartmann, R.;Kimmel, N.;Hajdu, J

Realising local government visions for developing district heating: experiences from a learning country

District heating (DH) has an important role to play in enabling cities to transition to low-carbon heating. Although schemes are commonplace in some countries, in ‘learning countries’ where building-level technologies make up the majority of heating systems there are numerous barriers to introducing DH. Local governments are seen as key actors in helping to create a ‘shared vision’ for DH amongst stakeholders. This study uses interviews with stakeholders from a range of sectors in the UK (an example of a learning country) to examine the visions of local actors for developing DH and the types of national policy that would support local implementation of these visions. The analysis shows that in engaging with DH development local governments seek multiple types of value. Realising this value will most likely happen by taking a long-term, planned approach to development. In contrast, national government policy is geared towards techno-economic criteria and may lead to only a minority of potential sites being developed, without realisation of wider social or environmental benefits aligned to local visions. The work highlights the importance of local strategic planning, enabled by aligned national policy, in realising the full economic, environmental and social benefits of DH

What happens in the Lab: Applying Midstream Modulation to Enhance Critical Reflection in the Laboratory

In response to widespread policy prescriptions for responsible innovation, social scientists and engineering ethicists, among others, have sought to engage natural scientists and engineers at the ‘midstream’: building interdisciplinary collaborations to integrate social and ethical considerations with research and development processes. Two ‘laboratory engagement studies’ have explored how applying the framework of midstream modulation could enhance the reflections of natural scientists on the socio-ethical context of their work. The results of these interdisciplinary collaborations confirm the utility of midstream modulation in encouraging both first- and second-order reflective learning. The potential for second-order reflective learning, in which underlying value systems become the object of reflection, is particularly significant with respect to addressing social responsibility in research practices. Midstream modulation served to render the socio-ethical context of research visible in the laboratory and helped enable research participants to more critically reflect on this broader context. While lab-based collaborations would benefit from being carried out in concert with activities at institutional and policy levels, midstream modulation could prove a valuable asset in the toolbox of interdisciplinary methods aimed at responsible innovation

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

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 wellcharacterized 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 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom 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.11Ysciescopu

Imaging single cells in a beam of live cyanobacteria with an X-ray laser

There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential

A data set from flash X-ray imaging of carboxysomes

Citation: Hantke, M. F., Hasse, D., Ekeberg, T., John, K., Svenda, M., Loh, D., . . . Maia, F. R. N. C. (2016). A data set from flash X-ray imaging of carboxysomes. Scientific Data, 3. doi:10.1038/sdata.2016.61Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere