Technical Highlight - September 2008
Short description: Nature Methods 5, 53-55 (2008)
Single particle electron cryomicroscopy (cryo-EM) is a powerful tool for structural examination of large multiprotein complexes. While it has become typical to obtain greater than 10 Å resolution structures of internally symmetric complexes, it has proven difficult to determine structures of asymmetric complexes at equally high resolution. Resolution limitations for asymmetric complexes can stem from structural heterogeneity in the sample and sample aggregation, or from the use of buffers that are incompatible with structure determination conditions. To overcome these problems, Kastner, Stark and colleagues have developed a sample preparation method called GraFix that can improve sample quality for structure determination by cryo-EM. GraFix combines the sedimentation of purified complexes in a density gradient (such as glycerol or sucrose) with a weak chemical fixation gradient. The fixation agent is added to the denser glycerol solution, and, once the sample gradient is established, the preparation can be fractionated. This treatment allows for the formation of chemically stabilized non-aggregated complexes suitable for negative stain cryo-EM, while avoiding artifact formation that may occur from direct addition of a chemical fixation reagent. Buffer exchange using a spin column can be used to remove glycerol from the fixed sample preparation to improve image contrast for unstained cryo-EM. The authors tested GraFix on several large macromolecular complexes, including the 70S ribosome and the anaphase promoting complex/cyclosome. In general, they observed fewer aggregated particles, more homogeneous samples, improved image contrast in negative-stained samples, and overall better image quality for statistical imaging analysis. The authors suggest that GraFix would be most useful in the structural determination of fragile or flexible complexes via cryoEM, and may even be appropriate for light scattering or X-ray diffraction techniques.
Berthold Kastner, Niels Fischer, Monika Mariola Golas, Bjoern Sander, Prakash Dube et al. GraFix: sample preparation for single-particle electron cryomicroscopy.
Nature Methods 5, 53-55 (2008). doi:10.1038/nmeth1139