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Not so cool

SBKB [doi:10.1038/sbkb.2011.54]
Technical Highlight - December 2011
Short description: A comparison of X-ray diffraction datasets obtained at ambient and cryogenic temperatures reveals temperature-dependent effects on structural models.

Small packing voids (red spheres) and alternative sidechain conformations are shown for the protein lysozyme. Image courtesy of James Fraser.

Cryogenic cooling of protein crystals has become standard practice for structural biologists, and the benefits are hard to argue against. Flash-frozen crystals transport easily to a synchrotron and are less susceptible to radiation damage, contributing to high-resolution structures of thousands of proteins. It is widely thought that cooling the crystals has minimal effects on the structural models, but several studies have indicated this procedure may introduce biases including protein shrinkage and reductions in local motions.

To re-examine this question, Alber and colleagues (PSI JCSG) compared structural models for 30 different proteins for which data were available at ambient and cryogenic temperatures. Using the programs qFit and Ringer to detect structural ensembles, the authors found that cryogenic cooling frequently remodels exposed and buried residues, increases lattice contact area, and eliminates certain functional micro-cavities. One of the major alterations is seen when comparing X-ray models with NMR data: in contrast to models from flash-frozen crystals, ambient-temperature crystallographic models show more conformations corresponding to flexible regions detected in solution using NMR. This was evident when examining X-ray and NMR data for the GTPase H-Ras. In H-Ras bound to a substrate analog, a catalytically incompetent conformation predominated electron density maps from both ambient and cryo-cooled conditions. However, at low electron density values often regarded as noise, the ambient-temperature data also displayed small populations of alternative conformations in segments previously found to be flexible in solution using NMR. These rare substates, including the catalytically competent conformation, were absent from the cyrogenic crystallographic model.

The authors propose that models from cryo-cooled crystals represent low energy, low entropy states that may be missing relevant alternative conformations. While these conformations can be discovered using Ringer or qFit on cryogenic data, ambient temperature data contain more information regarding conformational ensembles. As a result, they suggest that efforts be directed to ambient temperature data collection with emphasis on optimizing techniques that minimize radiation damage. This approach, coupled with new computational methods to define alternative conformations, would lead to structural models that are more informative and more representative of proteins in vivo.

Steve Mason

References

  1. J. Fraser et al. Accessing protein conformational ensembles using room-temperature X-ray crystallography.
    Proc Natl Acad Sci U S A. 108, 16247-16252 (2011). doi:10.1073/pnas.1111325108

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