X-ray Crystallographic Screening Frequently Asked Questions:
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What should I do to prepare for the crystal screen?
Before initiating a crystal screen, it is useful to establish “standard” soaking conditions to understand the tolerability of your crystal to ligands. Because this library is provided as a dry film, one can eliminate the need to test for co-solvent tolerance by not adding organic co-solvent. If your crystallization conditions permit, one may test soaking in cryo-solution to remove the added step of cryoprotection before freezing. In principle, ligands should diffuse into crystals very rapidly (minutes) but it can take longer to reach equilibrium for low solvent content crystals or partially occluded active sites. Zenobia typically soaks overnight, but this is a parameter that should be optimized for each crystal system. (back to top)
My crystals cracked when I added them to the soak, what should I do?
Crystal cracking can result from a few different scenarios. Typically, one wants to reduce the strain on the crystal by soaking at lower compound concentration for shorter time periods. Crystals may also be uniquely sensitive to certain compounds or compound classes. These compounds may bind at crystal contacts. In this case, gentler soaking conditions may be applied to these specific compounds.
If you have not tested soaking for a ligand known to bind to your binding site, the best path forward is to complete this experiment, even if it is a fragment of the ligand or substrate. This will suggest if ligand binding at the active site results in a conformational shift that results in crystal cracking.
In some cases, a ligand binding at previously unknown secondary binding sites can result in crystal cracking. If a subset of ligands results in crystal cracking and a co-crystal structure cannot be obtained by soaking, one may set-up more traditional co-crystallization experiments with the ligand to identify a new crystal packing. Ligand may be diluted out of the soaking solution or dry compound may be purchased from Zenobia for additional testing.
If a subset of mixtures results in crystal cracking, one may obtain individual compounds to deconvolute and identify the ligand resulting in the crystal crack. (back to top)
Some of the compounds are not soluble in my mother liquor. What should I do?
The crystal soak experiment establishes an equilibrium between free ligand and bound ligand. If some of the ligand is not fully dissolved, then the equilibrium includes undissolved ligand, dissolved ligand, and bound ligand. It has been demonstrated that this is not an issue in crystal screening. To ensure that your soak reaches full equilibrium including bound ligand, we suggest soaking these compounds overnight (if the crystal tolerates it). (back to top)
How can I use your plates to test different soaking conditions?
We suggest preparing the 20mM stock as described and completing serial dilutions of compound into a separate plate to test different soaking concentrations. (back to top)
What if I cannot obtain crystals in the absence of ligand? Can I still do a crystal screen?
Yes! In many cases, co-crystals may be obtained in the presence of a weakly binding ligand. One may soak this ligand out and soak a new ligand into the crystals. This can work even for very tightly binding ligands, but it may require very long soak times for the co-crystal in mother liquor containing no ligand.
Alternatively, Zenobia’s plates may also be used for co-crystallization experiments by adding protein and mother liquor directly to the drop and incubating as a typical sitting drop experiment. Compound concentration may be varied by serial dilution as described above. (back to top)
I have a hit, now what do I do?
This depends on the goal of your project. One may obtain additional dry powder to verify the hit using additional crystallography, an activity assay, or binding experiments. One may also look at the collection and the individual hits to design follow-on screening libraries to improve potency or specificity. Zenobia provides both the dry powder and design of follow-on libraries from this 288 plate collection. (back to top)
I didn't get any hits? How is this possible?
If this is a known druggable target, this issue may be technical. If you have not verified that a known ligand binds to your site, complete this experiment. If you do not have a known ligand:
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First, closely examine all of your electron density maps for a feature that is consistent but larger than a water molecule. It is possible that a cryo-molecule or crystallization reagent is binding to the ligand binding site and blocking binding of other ligands.
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Look closely at the crystal packing, is it possible that the active site is blocked by a neighboring molecule?
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If this is a “difficult” target, do you have a binding pocket that can accommodate a ligand? Is your target undruggable?
To verify a lack of hits, one may also set-up co-crystallization experiments.
Note that Zenobia will supply up to 1 hr of free consultation on projects that provide no hits to aid in troubleshooting. Your success is our success! (back to top)