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Technology Topics Crystallography

Some crystals like it hot

PSI-SGKB [doi:10.1038/th_psisgkb.2010.18]
Technical Highlight - May 2010
Short description: Having trouble crystallizing a protein from a thermophile? Try keeping your crystal plate warm.

Crystals of TM1030 in complexes with different DNA fragments.

Dinshaw Patel's team at Memorial-Sloan Kettering Cancer Center, New York, have published some remarkable papers over the past few years. Three papers in Nature have helped elucidate much of the mechanism of how the protein Argonaute plays a key role in gene silencing 1, 2, 3 . Part of the reason for their success is the use of the Thermus thermophilus version of Argonaute ... and growing crystals at 36°C.

Would this approach be generally useful for proteins from thermophiles — and what effect does the increased temperature have on the protein structure? That's a question Wladek Minor's group in collaboration with PSI MCSG asked 4 .

His team studied the crystallization of the transcriptional regulator protein TM1030 from Thermatoga maritima, an organism that grows best at 80°C. They set up crystallisation plates at 4, 20, 37 and 50°C, and at all these temperatures diffraction-quality crystals grew. Structures of crystals grown at 4, 37 and 50°C were solved, with and without DNA bound. The overall conformation at each temperature was very similar with an rmsd between the structures of only 0.2 to 0.4 Å. The mean B-factor decreased as the temperature increased, suggesting that TM1030 is more ordered at higher temperatures.

A search through the PDB gave an overall impression of the crystallization temperatures used, although the data are not comprehensive because early records usually omitted this information. The majority of protein crystallization data in the PDB reports crystallization temperatures of either 20 or 25°C, with a significant number at 4°C. Only 71 structures out of the 64,000 in the PDB have a crystallization temperature above 38°C; surprisingly, these include proteins from mesophilic organisms. Curiously, the PDB has 31 structures that apparently had a crystallization temperature above 100°C, although this looks to be an error as none of them had verifiable publications associated with them.

These results suggest that proteins can be crystallized at much higher temperatures than are typically tried. For the TM1030–DNA complex, the quality of the crystallographic data and refined structure are equally good, regardless of the temperature. If you're working on a protein form a thermophilic organism it is worth investigating higher temperatures — it's even worth a shot for proteins from mesophilic organisms.

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Maria Hodges

References

  1. Y. Wang et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.
    Nature 461, 754-761 (2009). doi:10.1038/nature08434

  2. Y. Wang et al. Structure of the guide-strand-containing argonaute silencing complex.
    Nature 456, 209-213 (2008). doi:10.1038/nature07315

  3. Y. Wang et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex.
    Nature 456, 921-926 (2008). doi:10.1038/nature07666

  4. K. D. Koclega, M. Chruszcz, M. D. Zimmerman, G. Bujacz, W. Minor et al. “Hot” molecular crystals.
    Cryst. Growth Design 10, 580-586 (2010). doi:10.1021/cg900971h

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