Assay Protocols

****The initial primary assay is an X-ray screen run against apo MurD ligase from Streptococcus agalactiae Uniprot Q8E186 and against MurE ligase form Escherichia coli K-12 MG1655. This will be carried out at Diamond light source.

Fragment screen:

Concentration of compounds: The XChem experiment requires very little compound at as high as possible concentration. We normally ask for 1 copy at 100 mM in DMSO. Lower compounds concentrations can also be used, but we are limited to the final concentration of DMSO that can added to the well that has the crystal in (in the case of MurE that is 10%). Thus the higher compound concentration, the higher the soaking concentration.
Volume of compound: The volume will differ based on the Echo plate used. The specs for the 384 ldv plate and the 1536 ldv plates are given at https://www.labcyte.com/products/consumables/echo-qualified-source-plates, however I would suggest using ~20 ul in the 384 ldv and ~5 ul in the 1536 ldv. The important thing is that the bottom of the well should be covered with a decent amount of compound for the Echo transfer to work.
Plate preparation: If you are not sending a full plate of compounds, try avoiding the outer most wells. This helps with potentials evaporation losses due to the film not sealing well. All compound plates should be sealed with a foil seal for 96 well plates. The plates lids are not sufficient.
Number of compounds we can test: In theory we can prepare up to 400 crystals per day at the DLS XChem facility; the exact number being dependent on the amount of beamtime available. The logistics in executing the XChem experiment (including driving from Oxford to Diamond and back, setting up the experiment, soaking and mounting) takes the same amount of days regardless of the amount of crystals involved. All data analysis are done with XChem Explorer (XCE) (http://tkrojer.github.io/XChemExplorer/) and Pan-Dataset Density Analysis (PanDDA) (https://pandda.bitbucket.io/strategies.html).