Dear colleagues,
A trimeric protein of 90 KDa in (25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 2 mM DTT) formed crystals at 18 degree in overnight with conditions having 4 or 8 % tacsimate pH 5.0, 12 % PEG 3350. I have freezed crystal in Cryo-solution of 20 % glycerol was diffracted to 3.2 but highly mosaic and smeared spots.
I thought of improve the crystal package so I have done at 4 degree to slow down the process with pre-cooled tips, reservoir solutions and plate. But again crystals formed in overnight with same morphology as before.
What to do to improve this crystals?
Attached crystals image...
You may wish to try microseeding, for doing so you crash the crystals, and make a seed stock, when setting new crystallization plates (with lower protein concentration) you can add a tiny amount of seed stock or dip a cat whisker (or equivalent) in the seed stock to inoculate it into the new crystallization plates
Those crystals look good!
I don't think they grow too fast. Overnight crystallization is reasonable.
I would try tweaking with the cryo here, or dehydration (see link).
http://scripts.iucr.org/cgi-bin/paper?S0907444905019451
the crystals in above image file was diffracted to 3.2 in ESRF BM14 beamline but only thing is that its mosaic and less spots for your reference in attachment.
Are you certain that the beam collimator is appropriately aligned? I saw comet spots in grad school that were caused from the micro beam collimator at CHESS being slightly out of alignment. Called the beam line scientist over and the comet tails were gone.
I've had some successes with improved diffraction with annealing. Try blocking the cryo stream for 1-2 seconds and then recollect. Also, if the protein binds something you may improve order with including the substrate in the cryo or co-crystallization. Furthermore, you can try to include glycerol in the initial crystallization to avoid the need for a cryo step. This may decrease the handling of the crystal. There are also additive screens through Hampton Research which might contained the perfect "wedge" to increase order.
Finally, try playing with the spot profile and size to see if you can decrease mosaicity and improve Rmerge of the dataset you already have. Best of luck.
thanks all for your sincere suggestions,
one thing is that this crystal was not carried in liquid nitrogen cane because in flight they wont allow to have liquid nitrogen. i just frozen the crystal with cryo and liquid nitrogen was completely dried while shipment the canes by flight.
From your diffraction images I can see that you have a cryo issue. The crystals can diffract much higher than what they did. I don't know from and to where you transfer your xtals and how else you could do it, but it is definitely a cryo issue.
Dear Nektaria Petronikolou,
i have transferred Xtal from india to france but while shipment the canes were kept in a cylinder without liquid nitrogen. in flight carrying the liquid nitrogen is not allowed ..
Yes, but if you fill up a dry shipping dewar with liquid nitrogen and then remove the excess liquid nitrogen, your xtals should remain frozen for a few days. There are a couple of things for you to try. First, make sure your dry shipping dewar works fine, and second freeze your crystals by soaking them in increasing cryoprotectant concentration. The latter has made a difference for me in the past. Good luck!
Since your crystals appear over night you can simply ship your frozen protein and grow the crystals on site. It is much easier to go through all possible cryo conditions and improve working conditions at the synchrotron compared to flying back and making new crystals every time.
thanks you all for your valuable suggestions. Next month i have beam line schedule. i will try with different cryo hope it will work.
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