In March this year, postdocs in Ivan Ahel’s group (Marion Schuller, Johannes Rack and Kang Zhu) cloned, purified and tested a range of SARS-CoV-2 macrodomain protein constructs. The best protein construct yielded, after optimisation of crystallisation conditions, crystals of the un-liganded enzyme that diffracted to very high resolution (1.0 Å) on beamline I03, allowing the structure to be rapidly determined and refined.
Crucially, these tests showed both that the crystals were tolerant to high DMSO concentrations, and that the active site (ADP-ribose binding) was empty and solvent accessible. Thus, an ideal crystallisation system for macrodomain fragment screening has been established in just a few weeks.
The XChem campaign and analysis were performed by Marion Schuller (Ahel group) and Daren Fearon, Alice Douangamath, Anthony Aimon and Victor Rangel (Frank von Delft's XChem team).
After growing a large number of crystals at Diamond and further optimisation of soaking conditions for adaptation to the screening process, the 1200-crystal experiment was completed within 72 hours, including all steps from fragment soaking, crystal harvesting and data collection on beamline I04-1 by the 22nd of May.
Analysis of the screening data using PanDDA (Nick Pearce and Conor Wild) for hit identification in the XChemExplorer interface (Tobias Krojer) revealed over 100 potential binding events which yielded a final total number of 80 hits (hit rate: 7%), which were refined and released on the 9th of July.
Following screening of the EU-OPENSCREEN fragment library (968 fragments at 100 mM designed by the EU-OPENSCREEN partner sites in collaboration with iNEXT-Discovery/Instruct-ERIC partners), a further 24 hits were identified and released on Fragalysis on the 16th of September, taking the total number to 124 (5.7%).
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