Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

The screening of smaller organic compounds called fragments using x-ray crystallography is an efficient method for identifying molecules that are starting points for drug discovery or biochemical tool compound development. Crystallographic fragment screening revealed not only the identity, but also where and how the fragments bind on the surface of the protein under study. This technique can be used on every biochemically or medically relevant protein target for which suitable protein crystals can be grown.

The quality of the results is intimately connected to the quality of the sample handling. We believe that the following visual demonstration will help researchers to carry out high quality experiments. Demonstrating the procedure will be Tatjana Barthel, a doctoral researcher and PhD student in my laboratory.

To begin, cut open the bag of the screening plate pre-warmed to room temperature, then remove the lid and the foil from the screening plate. Decant the five milliliter soaking solution in the reagent reservoir. Then fill each of the 96 reservoirs of the plate with 40 microliters of soaking solution using a 12 channel pipette.

Place the easy access frame on top of the screening plate and secure it with the included clamps by sliding them onto the left and right side of the device. Place the screening plate and easy access frame under the microscope and slide open the first well by moving the respective acrylic glass tile of the frame. Add 0.4 microliters of soaking solution from the reservoir to the fragment containing well using a fresh pipette tip.

Ensure that the drop covers the dried-on fragment. Place the crystallization plate that contains the largest crystals under the second microscope and cut open the sealing foil at one of the wells. Using an appropriately sized loop, transfer a crystal to the well of the screening plate under the first microscope.

Wash the loop in the prepared glass spot plate, then dry it by gently touching it to the tissue. Do this after every transfer to avoid cross-contamination. Use the microscope to ensure that the crystal has been properly placed.

Repeat the procedure for the second crystal. Move on to the next well, and repeat the procedure for all remaining wells. After crystals have been transferred to all 96 wells in the screening plate, remove the screening plate along with the easy access frame from under the microscope and place it onto the bench or table.

Then remove the easy access frame from the screening plate, seal the screening plate with sealing foil, and place it in the crystallization incubator or cupboard to incubate for the previously optimized soaking time. Two hours of incubation is sufficient, but overnight may be more convenient. Prepare the Unipuck foam Dewar with three Unipuck lids and half fill it with liquid nitrogen, keeping it on the ground.

Move the half-filled Dewar to the bench, and fill it completely to the very edge. Keep it filled to the upper edge during the entire experiment, and replace the liquid nitrogen frequently. Retrieve the screening plate from the incubator and remove its foil.

Place the easy access frame on top. Slide open the first well. Harvest one crystal from the drop and flash cool it in liquid nitrogen by plunging with a fast vertical movement.

Insert the sample in the proper puck position, and take relevant notes on the sample tracking sheet. Repeat the procedure for the second crystal. Go to the next well and repeat the positioning of other crystals until all three pucks are full.

Pre-cool the Unipuck bases in liquid nitrogen, and add them on top of the lids. Store the Unipucks in storage rack in a transport Dewar or storage Dewar. Keep them in liquid nitrogen at cryogenic temperatures until measurement.

Repeat the same procedure of harvesting and storing the crystals in Unipucks for the remaining samples in the screening plate. The variability of the crystal morphologies after performing the fragment soaking and crystal harvesting is shown here. Even crystals which look somewhat deteriorated were included, as they still result in useful data.

Data was collected at the beamlines 14.1 and 14.2 at the BESSY II synchrotron. Diffraction data collection was performed for each sample. The data was analyzed using FragMAXapp, focusing on the combination of XDSAPP for processing, fspipeline for structure refinement, and PenDA for hit finding.

This resulted in 15 hits on the AR protein complex using a DMSO free soaking condition. In a previous campaign of the F2X entry screen against the same target, including DMSO in the soaking condition, 20 hits were found. This means that 75%of the hits can be identified if DMSO is omitted.

For the success of the experiment, it is crucial that the crystals are handled properly and carefully during every step of the procedure. Crystallographic fragment screening has matured to a widely used technique, which is often applied in academia and in the pharmaceutical industry.