Hi Elizabeth, As suggested VMXi at Diamond may be able to help with in situ data collection without harvesting. I'd be happy to discuss if useful?
cheers Mike Sent from Outlook for Android<https://aka.ms/AAb9ysg> ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of CCP4BB <[email protected]> Sent: Thursday, November 23, 2023 7:04:45 AM To: [email protected] <[email protected]> Subject: Re: [ccp4bb] Fragile Crystals Hi Sorry if someone else has mentioned this already, but how about shooting your crystals in situ (i.e. while sitting in the plate)? I know that VMXi at Diamond can do this (in fact it's designed for his purpose) but haven't collected data in long enough not to know if it can be done elsewhere. Harry -- Dr Harry Powell On 22 Nov 2023, at 20:04, Phil Jeffrey <[email protected]<mailto:[email protected]>> wrote: Hello Morgan In addition to the other good suggestions, I have a few observations of my own. If your crystals crack without handling or adding anything to the drop, then they are extremely environment-sensitive. If that's the case, testing at room temperature will be problematic because that tends to be somewhat stressful on the crystal either mechanically (ye olde capillary mount method) or via dehydration (loop mounts with the sleeve). Growing in the presence of at least a little cryoprotectant as per Vaheh would be less stressful than multi-step processes like Tao-Hsin's advice unless your crystals can re-anneal after stress. Mounting directly from the drop is probably essential, and mounting under oil is a good thing to try in addition - apart from anything else oil on the drop slows down the environmental changes. Using Mitegen mounts might be less stressful on some crystals than standard nylon loops if they are mechanically sensitive. Spending some time optimizing the mechanics of your freezing technique might help significantly in reducing the amount of time your crystal dehydrates while moving through air. (Jim Pflugrath's article is full of useful information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461322/ ) Small crystals often freeze more smoothly than large ones - even for robust crystals like tetragonal lysozyme. Try a lot of crystals - I've had projects were two different crystals in the same loop from the same drop showed radically different diffraction. Also I've encountered several cases where the appearance of disorder varies within a crystal when using a microfocus synchrotron beam line (I mostly use FMX and AMX at NSLS2). Lastly, really cranky crystals rings a distant bell of something we encountered in the p19(INK4d)-Cdk6 structure back in the 1990's. I think it was Jie-Oh Lee that did the hard work on this, but in many instances crystals cracked in situ when merely opening the drop, and the fix was by adding a cross-linker to the well, resealing the drop and waiting for the cross-linker to diffuse: "The crystals were pretreated with glutaraldehyde (diffused into the drop from a reservoir of 30% glutaraldehyde) to reduce their tendency to crack and lose diffraction along b* and c*." https://www.nature.com/articles/26155#Sec9 Most crystals don't love being cross-linked, and I would call this a successful instance of a desperation maneuver. Good luck. Phil Jeffrey Princeton On 11/22/23 11:44 AM, Blake, Morgan Elizabeth wrote: Hello I am a PhD student working on a crystallography project to wrap up my dissertation research. I have purified a complex of two proteins, and I can consistently grow crystals in 10% PEG3350, 0.2M KSCN, 0.1M BIS-TRIS propane pH 7.5. These crystals have sharp edges and can grow to a large size (greater than 0.5 mm), but the crystals seem to be very fragile. When we open the drops to harvest the crystals, we have little time to harvest the crystals before they crack. When we move the crystals to a cryoprotectant, over time they start fracturing. We've tried using different percentages of glycerol, ethylene glycol, PEG400, and oil for cryoprotectants with no success. Needless to say, the crystals do not diffract well, with spot patterns that look very streaky/mosaic, which I presume is due to the defects that we see in harvesting/handling. We have screened for alternate crystallization conditions, but we seem to get the same morphology in other conditions. Does anyone have suggestions for additives we could use post-crystallization to help stabilize our crystals? 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