10 February 2020

Toward SAR by SFX

As our poll last year revealed, X-ray crystallography has inched out ligand-detected NMR to become the most popular fragment-finding method. One criticism often leveled at crystallography is biological relevance: very few proteins in nature are found in the crystalline state. Moreover, crystallography is a dish usually served cold, with crystals typically frozen in liquid nitrogen. The reason for this is that the powerful X-ray beams used to elucidate the structure of molecules also rip them apart, and freezing them slows the damage. But protein-ligand complexes at low temperature may not always reflect our more temperate world.

One approach to collecting crystallographic data at room temperature is to do so very quickly, before radiation damage can occur. This is done using serial femtosecond crystallography (SFX), in which many crystals are individually examined using brief, intense beams from X-ray free-electron lasers (XFELs). The X-ray pulses last less than 20 femtoseconds, a time so breathtakingly short that light only travels the width of a typical human cell. In a recent IUCrJ paper, Robin Owen, Michael Hough, and collaborators at the Diamond Light Source, the University of Essex, and elsewhere describe a high-throughput version.

The protein crystals themselves can be quite small, just 1-20 µm across, compared with the > 50 µm crystals typically used in crystallography. These microcrystals are mounted in silicon “chips” containing 25,600 little apertures; the X-ray beam can then be swept across each of the positions.

The researchers demonstrated that they could collect high-quality data for three proteins that are particularly sensitive to radiation damage: two heme peroxidases and a copper nitrite reductase. For all three proteins, they were able to determine high-quality structures of bound fragment-sized ligands. Indeed, some of the ligands were even smaller than all but the smallest fragments: imidazole (five non-hydrogen atoms) and nitrite (three non-hydrogen atoms). The latter case was particularly impressive given that the nitrite displaces a bound water molecule, so the difference between empty and liganded protein is even more subtle.

The first word of this blog is “Practical,” so how does this technique stack up? The researchers used 2-4 chips for each structure, and data collection took about 14 minutes per chip. Despite the miniaturization, sample consumption is not trivial: 1.4 to 6 mg of protein and 4-40 µmol of ligand for each data set. However, the researchers showed that could get by with less data – in some cases significantly so – and state that a 4-5-fold improvement in throughput would be straightforward. Using processing software such as PanDDa could further improve results. I suspect it is only a matter of time before we see the first FBLD by SFX screen. It will be fun to see how useful it turns out to be compared with established methods.

1 comment:

Matthew R. Lee said...

It will be interesting to see if and how such room temperature protein-fragment complexes end up differing meaningfully in the intermolecular distances, compared to the more routine low temperature crystal structures. More importantly, it will be helpful to understand whether or not the decision making for fragment-based design benefits from structural difference revealed through the room temperature SFX.