Novel Methods

Overview

Biomolecular SAXS signals can rapidly degrade due to radiation damage, so that flow or oscillating cells and large total sample volumes may be required. For particularly sensitive or hard to produce samples (such as of light sensitive proteins, metalloenzymes, and large complexes), and studies where multiple buffer conditions are probed (such as ligand binding experiments), sample consumption may be prohibitive.

We explored cryo-cooling of samples to temperatures of 100 K to prevent X-ray induced biomolecular disruption and aggregation during data collection. We identify SAXS-friendly cryoprotectant conditions that suppress ice formation upon cooling, and compare cryoSAXS profiles obtained in window-free variable-path-length cells with room temperature measurements for a variety of standard molecules. We obtain data sufficient for envelope reconstructions using scattering volumes as small as 100 nL, and find good agreement between cryoSAXS data and known atomic structures. We also developed low-volume fixed path-length sample holders for cryoSAXS. These enabled reproducible background subtraction without the scaling need by the windowed sample holders.

Cryo-cooled samples can withstand doses that are 2-3 orders of magnitude higher than typically used for SAXS at room temperature, comparable to those used in cryo-crystallography. While practical challenges remain, cryoSAXS opens the possibility of studies exploiting high brightness X-ray sources and mail-in high-throughput SAXS.

Papers

• Meisburger, S. P., Warkentin, M., Chen, H., Hopkins, J. B., Gillilan, R. E., Pollack, Thorne, R. E.(2013). Biophys. J. 104, 227-236.
• Hopkins, J. B., Katz, A. M., Meisburger, S. P., Warkentin, M. A., Thorne, R. E. & Pollack, L. (2015). J. Appl. Crystallogr. 48, 227–237.
  • The paper data is available.
  • A paper reprint is available.

Project Collaborators

• The Pollack lab at Cornell, particularly:
  • Steve Meisburger (now a postdoc at Cornell)
  • Andrea Katz (now a postdoc at Cornell)
• The Thorne group at Cornell, particularly
  • Matt Warkentin (former graduate student and postdoc)
• MacCHESS at the Cornell High Energy Synchrotron Source, particularly:
  • Richard Gillilan, beamline scientist