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?
Thanks for your advice!
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