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:
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.
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