Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source

The XChem Facility at Diamond Light Source provides routine, large scale crystal graphic fragment screening, supporting the entire crystal to deposition process. X-ray crystallography is an essential part to the FBDD toolkit. It is sensitive enough to identify weak binders and directly yields structural information about the interactions on a molecular level.

Fragment based drug discovery is a widely used strategy for lead discovery. It has delivered six drugs for clinical use and more than 50 molecules have been advanced to clinical trials. The impact of advances and efficiency provided by the XChem platform over traditional soaking methods, data collection and analysis methods best demonstrated visually.

Begin by choosing the crystals and compound location. Open TextRank from a PC and select the crystal tray, either from the list on the bottom right or by typing the barcode into the box at the top left. Select the correct image format and the single well view.

To add solvent or compounds to a drop without hitting the crystal, right click within the drop, but away from the crystal when a suitable crystal for an experiment is found while moving through the drop images. Open the Echo software and select new to dispense solutions using the acoustic dispenser. Choose the correct source well plate and the liquid class.

Ensure the correct plate type is selected as the destination plate. Then check the custom box and continue. Select import, and choose the relevant batch file.

Then complete the import steps as prompted by the software. Use the plate maps to check the solution to be dispensed and the destination locations. Then run the protocol following the prompts as they come up.

The solutions from the source plate will dispense into the chosen crystal drops. Store the plate in the incubator for the required time. To harvest the crystals using the semi-automatic crystal harvesting device, press the start workflow button to move to the first selected well position.

If the crystal has survived, mount it in the loop and plunge it into the liquid nitrogen, placing it in position one in the first puck in the list. Select the appropriate description for the crystal from the interface. If the drop is a compound soak, record the description of the compound state.

If the crystal has been successfully mounted, select mounted, otherwise, select fail. Once the crystals have been harvested, take the pucks to the barcode scanner and place them in the holder one at a time to scan the puck and pin barcodes. Once the scanning is completed, place the lids on the pucks and store them in a liquid nitrogen storage dewar.

To recollect the miscentered samples, look at the sample changer view in ISPyB and select rank by AP resolution to grade the samples by auto processed resolution in a color graduation from green to red. Click on the samples to check for any red or yellow samples. Then check the crystal snapshots to see whether the crystal has been centered.

To retrieve and analyze Diamond's auto processing results through XChem Explorer or XCE, in a terminal go to the sub-folder processing and use the alias XCE to open XChem Explorer. Select the update tables from data source button in the overview tab to refresh the summary of the experimental data. Under the settings tab, select the data collection directory, open the datasets tab.

Choose the target from select target dropdown menu, select get new results from auto processing from the datasets dropdown menu and click run. To calculate initial maps using Dimple, open the maps tab, choose the reference model from the dropdown menu and select the desired datasets followed by run Dimple on selected MTZ files. To generate ligand restraints, select the desired datasets followed by create SCF or PDB or PNG file of selected compounds from the maps and restraints drop down.

To identify hits using Panda, select the Pandas tab, ensure the output directory is correctly defined and run panda. analyze from hit identification dropdown menu. To analyze hits identified by Panda, run panda.

inspect from the hit identification dropdown menu. To open Coot with the Panda control panel. Load average and 2mFo-DFc maps from Dimple for comparison with the event map and model.

Once a ligand has been fitted, click on merge ligand with model and save model before navigating to another event to avoid losing any changes to the bound state model. Annotate the binding event using the event comment field and annotate the binding sites using record site information. Once all viable ligands have been modeled, merged, and saved based on the event map, close panda.inspect.

Export the Panda inspect models back into the project directory and launch an initial round of refinement for the selected datasets, and the refinement will now be visible in the refinement tab. The XChem pipeline for fragment screening by X-ray crystallography has been extensively streamlined, enabling its uptake by the scientific community. This chart demonstrates the uptake and consolidation of the user program from 2015 through to 2019 with the creation of block allocation groups in 2019 and the resilience of the platform through the COVID-19 pandemic in 2020.

Successful campaigns yield a three dimensional map of potential interaction sites on the target protein. A typical outcome is the XChem screen of the main protease of SARS-CoV-2. The known sites of interest such as enzyme active sites and sub pockets are shown in yellow.

The putative allosteric sites such as those involved in protein-protein interactions are shown in magenta and crystal packing interfaces generally considered false positives are shown in green. Historically, using crystallography as a primary fragment screen has been difficult. This study documented the XChem pipeline protocols from sample preparation to the final structures.

Crystallographic fragment screening compliments other biophysical techniques and is usually essential for progressing fragment hits to lead compounds. It can be applied to any drug discovery target class.