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]> 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? >> Thanks for your advice! >> ------------------------------------------------------------------------ >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> <https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C01%7Cpjeffrey%40PRINCETON.EDU%7C7df53dda9c18474a25d208dbeb7bbeec%7C2ff601167431425db5af077d7791bda4%7C0%7C0%7C638362688963366377%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=tru7KwHbWYoYZZ3AOrMUilKXqAopNBpCZ32XASFWcaQ%3D&reserved=0> > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing > list hosted by www.jiscmail.ac.uk, terms & conditions are available at > https://www.jiscmail.ac.uk/policyandsecurity/ ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
