SAR by MicroED?

Of the dozen-plus methods to find and characterize fragments, only two have historically been able to provide detailed binding information: protein-detected NMR and X-ray crystallography. Earlier this year we described how researchers at Astex are using cryo-EM for FBLD. In a new open-access Communications Biology paper, Hongyi Xu and collaborators at Stockholm University, Lund University, and SciLifeLab describe another variation of cryo-EM.

Microcrystal electron diffraction (MicroED) is something of a cross between standard crystallography and cryo-EM. Like more “conventional” cryo-EM, an electron microscope is used to collect data on flash-frozen samples. But rather than painstakingly reconstructing thousands of images of individual protein molecules, MicroED uses diffraction of electrons from crystals that are far too small for standard X-ray crystallography. Even though electrons rather than photons are being scattered, diffraction is diffraction, so well-established X-ray crystallography methods can be used for processing MicroED data.

The researchers focused on human carbonic anhydrase II (HCA II), a popular model protein that has also been used to showcase X-ray crystallographic, native MS, and SPR methods. The microcrystals were less than 500 nm thick, smaller than most bacteria and at least 100-times smaller than typically used for X-ray crystallography. MicroED data were collected on microcrystals of native HCA II as well as microcrystals that had been soaked for 20 minutes with the known ligand acetazolamide.  At just 13 heavy atoms, this approved drug is still comfortably a fragment.

Data were collected to 2.5 Å resolution, which is modest especially compared with the 1.1 Å resolution of a published crystal structure. Nonetheless, the ligand density was clearly visible, and the refined model was very similar to the published crystal structure as well as another published structure of the complex determined by neutron diffraction. The researchers note that the observed features are similar to those that could be expected of a crystal structure solved at the same resolution.

The researchers note several potential advantages of MicroEM over X-ray crystallography. It can sometimes be difficult to obtain sufficiently large crystals for crystallography, particularly when protein is limited. Smaller crystals may allow faster diffusion of ligands into the crystals. And at higher resolution, individual hydrogen atoms are more easily resolved using electron diffraction than X-ray diffraction.

Will these advantages be enough to make MicroED a truly practical method for FBLD? It is not clear from the paper how long the data collection and processing took, HCA II is a friendly protein to work with, and acetazolamide is a high affinity ligand. That said, MicroEM was first described only in 2013, and this paper demonstrates that ligand binding modes can be determined. It will be fun to watch this technique develop.