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Technology Topics Reagents

Greasing the Path for SFX

SBKB [doi:10.1038/sbkb.2014.241]
Technical Highlight - January 2015
Short description: Grease is an ideal microcrystal carrier medium for serial femtosecond crystallography.

A simple three-step process prepares microcrystals suspended in grease for SFX. 1

X-ray free-electron laser (XFEL) technology is enabling novel ways to tackle challenging structural biology questions. One such approach is serial femtosecond crystallography (SFX), a technique that enables atomic-resolution structure determination from tiny, micron-sized crystals. In this technique, a stream of microcrystals is flowed to intersect with the ultrabright femtosecond pulses of an XFEL. Though the XFEL pulse annihilates each crystal, it is so fast that a diffraction pattern can be collected before radiation damage occurs. Thus, SFX has the potential to solve the structure of proteins that resist forming the large crystals needed for traditional X-ray crystallography.

The SFX technique requires that tens of thousands of diffraction snapshots be captured from microcrystals in random orientations. Several methods to stream microcrystals into the path of the XFEL beam have been described, but the difficulty is in getting the crystals to actually intersect with the femtosecond pulses. The most common approach, using a liquid microjet, has poor efficiency. Alternatively, microcrystals can be suspended in a viscous medium such as lipidic cubic phase to better control flow rate, but to date, crystallization in this medium this has been limited to membrane proteins.

Sugahara and colleagues now report a simple yet versatile and efficient method to put microcrystals into the path of an XFEL beam. They identified a mineral oil–based grease in which microcrystals can be suspended simply by mixing the microcrystal slurry with the grease on a plate. The mixture is inserted into a pipet tip and then loaded into a syringe. By selecting an optimally sized syringe needle and extrusion rate, the authors were able to flow the microcrystal-containing grease in a stable, continuous column into the focal spot of an XFEL.

In work performed at the SPring-8 Angstrom Compact Free Electron Laser facility in Japan, the authors demonstrated their method by solving high-resolution (2-Å or finer) structures from microcrystalline samples of lysozyme (PDB 3WUL), glucose isomerase (PDB 4W4Q), thaumatin (PDB 3WXS) and fatty acid–binding protein type 3 (PDB 3WXQ). Because the flow rate can be carefully controlled, compared to a liquid microjet, sample consumption was reduced and the hit rate of crystals intersecting with XFEL pulses was improved. It remains to be seen how this method will perform in the hands of others, but the data so far suggest that it should be broadly applicable.

Allison Doerr

References

  1. M. Sugahara et al. Grease matrix as a versatile carrier of proteins for serial crystallography.
    Nat Methods. (10 November 2014). doi:10.1038/nmeth.3172

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