Extensive Angular Sampling Enables the Sensitive Localization of Macromolecules in Electron Tomograms

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect macromolecules in tomograms, template matching (TM) is often used, which uses 3D models that are often reliable for substantial parts of the macromolecules. However, the extent of rotational searches in particle detection has not been investigated due to computational limitations. Here, we provide a GPU implementation of TM as part of the PyTOM software package, which drastically speeds up the orientational search and allows for sampling beyond the Crowther criterion within a feasible timeframe. We quantify the improvements in sensitivity and false-discovery rate for the examples of ribosome identification and detection. Sampling at the Crowther criterion, which was effectively impossible with CPU implementations due to the extensive computation times, allows for automated extraction with high sensitivity. Consequently, we also show that an extensive angular sample renders 3D TM sensitive to the local alignment of tilt series and damage induced by focused ion beam milling. With this new release of PyTOM, we focused on integration with other software packages that support more refined subtomogram-averaging workflows. The automated classification of ribosomes by TM with appropriate angular sampling on locally corrected tomograms has a sufficiently low false-discovery rate, allowing for it to be directly used for high-resolution averaging and adequate sensitivity to reveal polysome organization

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

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

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

Three-dimensional reconstruction of the giant mimivirus particle with an X-ray free-electron laser

10.1103/PhysRevLett.114.098102Physical Review Letters114909810

Intergenerational impacts of maternal mortality: Qualitative findings from rural Malawi

Background: Maternal mortality, although largely preventable, remains unacceptably high in developing countries such as Malawi and creates a number of intergenerational impacts. Few studies have investigated the far-reaching impacts of maternal death beyond infant survival. This study demonstrates the short- and long-term impacts of maternal death on children, families, and the community in order to raise awareness of the true costs of maternal mortality and poor maternal health care in Neno, a rural and remote district in Malawi. Methods: Qualitative in-depth interviews were conducted to assess the impact of maternal mortality on child, family, and community well-being. We conducted 20 key informant interviews, 20 stakeholder interviews, and six sex-stratified focus group discussions in the seven health centers that cover the district. Transcripts were translated, coded, and analyzed in NVivo 10. Results: Participants noted a number of far-reaching impacts on orphaned children, their new caretakers, and extended families following a maternal death. Female relatives typically took on caregiving responsibilities for orphaned children, regardless of the accompanying financial hardship and frequent lack of familial or governmental support. Maternal death exacerbated children’s vulnerabilities to long-term health and social impacts related to nutrition, education, employment, early partnership, pregnancy, and caretaking. Impacts were particularly salient for female children who were often forced to take on the majority of the household responsibilities. Participants cited a number of barriers to accessing quality child health care or support services, and many were unaware of programming available to assist them in raising orphaned children or how to access these services. Conclusions: In order to both reduce preventable maternal mortality and diminish the impacts on children, extended families, and communities, our findings highlight the importance of financing and implementing universal access to emergency obstetric and neonatal care, and contraception, as well as social protection programs, including among remote populations

Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency