Crystal-based fragment screening is a straightforward process at FragMAX. However, project success mostly depends on the characteristics of the crystal system used for screening. It is therefore important that the system is ready before you consider applying at FragMAX because we cannot easily extend approved proposals that fail due to technical problems. The list below gives an overview of some of the most important points to consider, and please do not hesitate to get in touch if you have any questions!

Crystallographic fragment screening requires several hundred protein crystals (for fragment soaks and for establishing tolerance to organic solvents). It is therefore of utmost importance that crystals can be grown reliably and reproducibly in large quantities without needing to prepare tens of crystallization plates. Large quantities mean at least two hundred crystallization droplet with suitable crystals.

Crystal preparation at FragMAX is optimized for 3-lens SWISSCI sitting-drop crystallization plates with typical drop volumes ranging from 100 – 400 nL. We are not able to support experiments on-site that require crystallization in hanging-drop plates, 24/48-well sitting drop, or LCP plates. Nevertheless, we are happy to look for workaround wherever possible.

We can grow crystals at 4 and 20 degrees at FragMAX, but the entire crystal preparation process can currently only be done at room temperature. It is often possible to soak and mount crystal that were grown at 4 degrees at ambient temperatures, but of course not in all cases. Please test in advance if your crystals only grow at 4 degrees and if they can tolerate being put at room temperature for prolonged periods of time without deteriorating.

Crystals should be robust and big(ish), i.e. larger than 50 μm in at least one direction. While both criteria are not entirely objective, it is obvious to most practitioners that all stages of the experiments are simpler with easy-to-handle crystals that do not fall into pieces at first sight and are of reasonable size.

Fragments are applied through crystal soaking at FragMAX. Hence, if you know the “site of interest” of your protein, please check that this region is accessible through solvent channels and is not blocked by crystal contacts.

It is important that crystals diffract uniformly and reliably to resolutions below 2Å (ideally), but at least up to 2.8Å. Better data quality simply makes ligand identification easier, but it also increases the amount of information that be obtained from the structures.

The compounds from all our fragment libraries are dissolved in DMSO and the FragMAXlib library is additionally dissolved in Ethylene glycol. In a typical experiment, we would prepare a 10 – 30 % solution of each fragment with crystallization buffer and add it to the crystallization drop. Hence, crystals should show some tolerance towards these solvents and if your crystal system can cope with higher solvent concentrations, you are able to soak with higher ligand concentrations which should in principle help to identify lower affinity fragments bound to the protein. Additionally, the added solvent, combined with the low volume of crystallization solution around our loops often obviates the need for additional cryo protection. However, we are in principle able to dissolve the FragMAXlib in your crystallization buffer if your crystal system cannot tolerate either DMSO or Ethylene glycol. And we can also add an extra cryo-protection step to our crystal preparation workflow in case this is necessary. But regardless, we advise to establish the tolerance to DMSO and Ethylene glycol beforehand and to check if solvent molecules are occupying interesting binding sites.

Understand your crystal system and be aware of complicating issues like twinning, pseudo-translations/ symmetry, multiple crystal forms in the same crystallization drop or susceptibility to (site-specific) radiation damage.

We understand that science is rarely predictable, that sometimes unorthodox solutions are required and that there are many possibilities that we have not yet considered. Therefore, don’t hesitate to get in touch if there is anything you’d like to discuss, even if it does not fit the “standard” FragMAX workflow.