Crystallisation and Sample Preparation

With standard UDC parameters, approximately 250 samples are collected per shift.

Tier 1 - preliminary experiements and solvent testing - 1 shift

Tier 2 - Full library screening - 4 shifts

Tier 3 - Follow up compounds (varies based on number of ligands)

Potential Cause:  Are you using a different batch of protein to previous plates?

Corrective action(s):

• Definitely try increasing protein concentration and decreasing precipitant - might need a lot more than you expect!  (e.g. for protein 8->30mg/ml, take PEG 25->3%.)  Go look again carefully at basic crystallization theory and the phase diagram.
• Definitely do seeding, seeding helps with so many problems, we'll mention it a lot, if you aren't familiar with it see details by Douglas Instruments on seed stocks (link).
• Look carefully at your purification protocol - is it really yielding consistent protein quality?  Be sure gel filtration is the final step.
• Try additives (e.g. arginine) or detergent
• If none of the above work you will have to optimise your crystallisation condition again and check diffraction quality, screens with very broad ranges of conditions (pH, salt, etc)

Possible Causes:  Was your original crystallisation optimised for larger, hanging drop format?

Corrective action(s):

• Conditions can change a LOT between formats: get SwissCi plates and optimise aggressively - all the things in 'Crystals are not growing reproducibly' section, above
• The good news: you know the protein likes to crystallise, so it's unlikely to be format specific - keep looking.
• Take seeds from the hanging-drop crystals, and seed the sitting drop conditions.
• We will soon support other sitting drop plate formats (it's taking time for complicated but boring reasons)
• We have harvesting protocols that allow you to pick crystals from hanging drop slides and transfer them to the compounds pre-dispensed in SwissCi plates.  It is significantly slower, though, and crystals need to like it, so we suggest it to be last resort.
• Protein engineering may be necessary:  SER, crystallization epitopes, different construct lengths, crystallization chaperones... obviously a lot more work.

Possible Causes:  Your crystal system may be sensitive to DMSO.

Corrective action(s):

• Do a more extensive solvent tolerance test
• Try even lower DMSO concentrations: the first priority is that the crystals survival;  maximising compound concentration is much less important.
• Try different solvents.  Ethylene glycol has been effective for several systems and several of our libraries are available prepared in EG now.  We have also had some interesting preliminary results using hydrotropes.
• Try very short soaks - one user's system required <5secs.

As a last resort:  If you need to do short soaks the Shifter has a Dip-Soak programe that makes this method easy to do and enables you to add the compound straight before harvesting to reduce damage to your crystal.  We would prefer if you optimise your crystallisation conditions to be compatible with a longer soak in DMSO or EG but if this is the only way we can do it!

Possible Causes:  Some proteins are like that...

Corrective action(s):

• If resolution is not affected, you don't have a problem: during processing, crystal forms can be clustered retroactively and handled individually.
• Mount a few dozen crystals, and carefuly compare outcomes to morphology: it may be visible after all.
• Explore more cryoprotection protocols: since the crystal packing evidently lies near a phase transition, certain prototols might drive the system to a particular phase.  See #4 above.
• Do more aggressive optimisation as in #3 - don't forget seeding with the best crystal form!

Possible Causes:  Crystallisation condition may contain a volatile solvent or high salt.

Corrective action(s):

• Place filter paper soaked with some solvent (e.g. iso-propanol, water) place around the shifter mounting aperture.
• Add a small volume of reservoir solution to all drops before transferring compounds
• Search for easier crystallisation conditions
• Talk to us about using the Watershed (details can be viewed in the XChem Pipeline subpage)

Possible cause:  Are they salt crystals?

Corrective action:

• If they are salt then we recommend searching for a low-salt crystallisation condition

Possible causes:  If they are protein crystals then your crystallisation condition remains meta-stable even after crystals have grown.

Corrective action(s):

• Optimise your condition to find a lower-saturation condition - try seeding!

• Try adding reservoir solution to drops before transferring compounds.

• See if using the Watershed helps, it may help with this but we can't guarantee it, details of the Watershed can be viewed in the XChem Pipeline subpage

Last resort:  Characterise how few droplets you can have open at once, by seeing how quickly the showers of crystals form.  Once we know, we can modify the soak/harvest workflow accordingly.  It makes everything a lot more tedious, though, so please try optimisation first.

Possible Causes:  It always grew like this.

Corrective action(s):

• Change your crystallisation volumes. For instance, a system which showed skin when grown from 500 nL + 500 nL drops stopped showing skin when grown from 200 nL + 200 nL (a preferred volume, anyway).

Possible Causes:  Some crystals are like that...

Corrective action(s):

• If they are large, chunky needles i.e. larger than our beam (60x50 um), then you should be ok

• If they are thin needles try to get them to lie consistently across the centre of the loop during harvesting (tough!)

• You may need to do assisted data collection: it requires you watching every crystal and fixing any mis-centring - it's a bit tedious but we rigged the software to make it very easy.

• We're working with Mitegen to develop new needle-supporting loops.

• We're always working on various ways to improve data collection for these very cases, some things may take a long time to impelment but it's the nature of these big projects and updates

Possible Cause:  Is your crystallisation condition straight from a screen?

Corrective action:  Do more optimisation

Possible Cause:  How extensively have you tried optimisation?

Corrective action(s):  

• Try many more cryo-protection protocols
• Try quick-dip-and-freeze
• Gradual increase in cryoprotectant (annoying but supportable)
• Don't forget sugars!

Potential Cause:  Crystals may age faster than you realise

Corrective Action(s):  

• Check the diffraction of freshly grown crystals and prepare fresh crystals before you visit Diamond

Possible Causes:  Your crystals may be growing as sub-microscopic clusters or stacks of plates.

Corrective action(s):

• Your system needs more optimisation
• Try seeding (!!)
• If data quality from processing is okay, then you may not have a problem:  modern data collection and processing lets you get away with murder, and besides, data don't actually need to be particularly accurate (it's not SAD phasing!)

Potential Cause:  If the bond is not physiolopgically relevant then your protein may be sensitive to oxidation

Corrective Action(s):

• Add a redox reagent (DTT, TCEP) to during your sample preparation and crystallisation
• We can also add a redox reagent to crystallisation droplets using the ECHO before transferring the compounds