Crystallographic vs computational fragment screening

Several recent Practical Fragments posts have touched on crystallographic screening: from ultra-high concentration screening of “MiniFrags,” to an extensive analysis of fragment structures in the protein data bank, to an open-source effort to develop new antibiotics. A new paper in Phil. Trans. R. Soc. A by Tom Blundell and collaborators at University of Cambridge, the Diamond Light Source, University of Oxford, and several other institutes provides a useful synthesis and an interesting comparison with computational approaches.

The researchers were interested in the bacterial protein PurC, also known as SAICAR synthetase, which is essential for purine biosynthesis and is sufficiently different from its human orthologue to be an attractive antimicrobial target. The protein has an extended binding site that can accommodate ATP as well as its substrate CAIR and an aspartic acid. Using a traditional screening cascade, 960 fragments were screened at 5 mM in a thermal shift assay, resulting in 43 hits. Each hit was then soaked at 10 mM into crystals of PurC, resulting in 8 bound structures, all of which occupy the ATP-binding pocket. Isothermal titration calorimetry revealed dissociation constants as good as 178 µM, with a ligand efficiency of 0.39 kcal/mol/atom.

Next, the researchers ran a computational screen, Fragment Hotspot Maps. This confirmed the main fragment-binding site. Indeed, the crystallographically-identified fragments even make the hydrogen-bonding interactions predicted by the model. However, the computational approach also identified three other hot spots, two in the active cleft and one on the rear of the protein. There was also a “warm spot” next to the ATP-binding site. Are these real, or computational artifacts?

To address this question, the researchers screened fragments at a much higher concentration at XChem, and processed the data using the PanDDA software we’ve previously described. They screened two libraries of fragments at 30-50 mM: 125 “shapely” fragments and 768 “poised” fragments designed for rapid follow-up chemistry. The 8 hits from the first crystallographic fragment screen were also included. This exercise yielded structures for 35 fragments, 60% of which bound in the ATP-binding site, including all 8 of the previously identified ones. Most of the other fragments bound in shallow pockets or near crystallographic interfaces; only one of the other hot spots predicted computationally had a bound fragment, and that was present at low occupancy. Some hits made new interactions around the ATP-binding site, but none bound in the predicted warm spot. Unfortunately, the proportions of fragment hits coming from the two libraries are not broken out.

So in summary, both computational and crystallographic screening correctly identified the “hottest” hot spot, but each approach also identified additional sites that were not confirmed by the other. The researchers ask, “are these sites truly hot spots… or are they weak binding sites routinely seen in crystals?”

This is indeed the key question, and it would be interesting to see whether other computational approaches – such as FTMap or SWISH – are able to shed light on the matter.