A new publication is available that describes practical aspects of collecting High-throughput SAXS data at the SIBYLS beamline, with a focus on challenging low concentration samples. Additional practical advice is laid out with respect to interpreting the resulting data.
Dyer KN, Hammel M, Rambo RP, Tsutakawa SE, Rodic I, Classen S, Tainer JA, Hura GL. “High-throughput SAXS for the characterization of biomolecules in solution: a practical approach.” Methods Mol. Biol. 2014;1091:245-58. link
The SIBYLS beamline was instrumental in providing key structural data for two recent publications exploring the dynamic nature of DNA repair.
The Mre11‐Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP‐driven states regulate the sensing, processing and signaling of DNA double‐strand breaks are largely unknown. Here we design structure‐based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP‐driven movements within the catalytic domains. With this strategy we identify Rad50 separation‐of‐function mutants that either promote or destabilize the ATP‐bound state. Crystal structures, X‐ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP‐induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis‐induced opening is essential for DNA resection. Reducing the stability of the ATP‐bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double‐strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11‐Rad50‐Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure‐based mutational analyses. These collective results suggest that ATP‐dependent Rad50 conformations switch the Mre11‐Rad50 complex between DNA tethering, ATM signaling, and 5′ strand resection, revealing molecular mechanisms regulating responses to DNA double‐strand breaks.
read more in the full articles…
Young researchers are encouraged to apply for the Cancun conference “Dynamic Structures in DNA Damage Responses and Cancer” from the 12-15th February, 2014. We expect that there will be lively discussions on both methods and results during the sessions and good opportunities to interact with top colleagues. To inspire your productive discussions the conference venue has breath taking views of the Caribbean Sea and a great beach to enjoy with colleagues. There are a few spots to join the conference and some for late breaking talks so if you or your group members would appreciate this intimate and informative meeting on dynamic structures in DNA damage responses and cancer, then we would encourage you to APPLY. We are putting together many of the people driving advances under one roof to make this a meeting that will prove uniquely productive and informative for those working in this area and seeking collaborators. Although you may have a busy schedule we aim to make this meeting worth your taking the time to participate by directly aiding research progress and collaborations.
Greg Hura and colleagues developed and applied nanogold labels for DNA complexes with proteins examined by small angle X-ray scattering (SAXS) to follow DNA conformations acting in error detection by the mismatch repair (MMR) system in solution. This technique can examine short or long pieces of DNA and in most solution conditions, including those closest to cellular environments. This technique is expected to be useful for many biologically important systems involving DNA complexes and conformations. In this manuscript the authors reveal DNA bending followed by straightening by the repair protein MutS at the site of a mismatch as a suitable mechanism for error detection and signaling needed to avoid mutations and cancers and to control microbial stability and evolution in response to environmental stress.
Mismatch DNA bending by MutS and straightening in the presence of ATP. Contour plots of the distribution of DNA ends are visualized by placing the structural information from the crystal structure of MutS/DNA on the same scale as the distance and pop- ulation information from the P(Dij) distributions. The P(Dij) distributions from 71-bp DNA in the presence of MutS (left) and the presence of MutS and excess ATP (right) set contour levels. The widest part of the distribution is the width of the gold nanocrystal. DNA of the crystal structure has been ex- tended to 71 bp for the MutS/DNA complex and replaced by straight DNA for the ATP model.
For all the details please check out the full manuscript:
We are pleased to announce the 4th annual SIBYLS bioSAXS workshop.
Date: October 8-9, 2013 Location: Advance Light Source (ALS) at Lawrence Berkeley National Laboratory , Berkeley, CA
The SIBYLS team will host a workshop with strong emphasis on experimental aspects of Small Angle X-ray Scattering techniques in structural biology. The two-day workshop will provide training on experimental techniques and software tutorial sessions primarily for biological SAXS studies. The latest advances in SAXS studies on biological systems will be reported and discussed by invited experts including our keynote speakers Prof. Peter Moore (Yale), Pau Bernando (CNRS France) and John Tainer (Scripps). Also planned are presentations on solution structure modeling techniques for proteins, RNA, DNA-protein complexes. The second day of the workshop will be dedicated for processing of workshop participant data previously collected at SIBYLS.
Participants will receive updates on current software dedicated to analyze SAXS for structural biology:
Enrollment is limited to 30 participants.
Several new crystal structures of the 70S ribosome in complex with EFG and non-hydrolyzable GTP analogs have revealed how the ribosome directionally translocates mRNA and the tRNAs through the A, P, and E sites and how specific features of EFG and ribosomal RNA act as pawls to enforce this ratcheting mechanism. The new structures were solved in the Cate, Noller, and Ramakrishnan labs and were published in the June 28 edition of Science. Many of the new structures were made possible by data collected at the SIBYLS beamline.
If you want the boiled down version of these new results then read this succinct comment by Marina Rodnina.
A figure liberally lifted from her comment.