for cryoprotection just drag it through paratone-N (put next to your crystal drop) and you dont have to worry about ill effects on the crystal (usually). this will cut down the long road with cryoprotectant search to minimal. (..not sure if this had anything to do with the original topic, but since it keeps coming up...) it doesnt penetrate the crystal and works and you dont have to waste time and crystals on finding a solution with some soluble cryoprotection additive that doenst kill the crystal..
tommi Quoting Leonard Thomas <[EMAIL PROTECTED]>: > The first thing to try before going down the long road of fussing > with cryo is to take a shot at room temp. and see how your crystal > diffracts in general. It is true it may be a cryo problem, but if > the non cryo protected crystals do not diffract then why would one > expect the cryo protected one to. > > As for controlling crystal growth. I would second what Shane wrote > and try seeding. Also trying the usually additives and varying > protein concentration/precipitant concentration should help also so. > > Len > > > On Apr 4, 2007, at 8:56 AM, Shane Atwell wrote: > > > Streak seeding all your trays should give you a better handle on > > nucleation. You might be too high in protein or precipitant w/o the > > seeding, hence the showering and rare nice crystals. > > > > Varying cryos, or cryo concentrations, or how the cryo is added can > > help > > a lot. Try 5 or 6 different cryos at 3 concentrations each. When you > > have the best nailed down then try sequential transfers of the > > crystals > > from low to target concentrations (e.g. into 5% for a couple minutes, > > then 10, 20, 25%). Also, you can try growing the crystals w/ a bit > > (5%) > > or the final conc or cryo already present. Should help in getting them > > habituated to the cryo. > > > > Shane Atwell > > > >> -----Original Message----- > >> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On > >> Behalf Of Jenny > >> Sent: Wednesday, April 04, 2007 5:50 AM > >> To: [email protected] > >> Subject: [ccp4bb] bigger size - > better diffraction? > >> > >> Hi, All, > >> > >> I got a crystal that diffracts at 3.3A in house.The crystal > >> size is about 0.2mm* 0.1mm * 0.2mm. At first I thought the > >> size is fine,but it turns out the smaller ones diffract > >> worse.I guess the reason is that > >> the cell unit is really big (126.292 126.292 134.904 p4212, > >> pretty big for a 10kD protein, isn't it?) > >> > >> So looks like I need to grow bigger crystals in order to get > >> better diffractions.The problems is ,every time when I set up > >> trays, the growing conditions is not exactly the same, so I > >> have to set up a whole tray or maybe even 2 trays , then 2 or > >> 3 conditions will jump out with good crystals ( 2 or 3 > >> nucleation site ) and some of the others will show lots lots > >> of small crystals.I used NaCl as the salt, in a 4*6 tray, the > >> [NaCl] is going from 2.0,2.05,2.1,2.15,....something like > >> that and 0.05M does make big difference.I used Urea as the > >> additive in this case ( 25 m ~ 100 mM) and tried > >> 2+2,3+1,3+2, 3+1 ( 3 uL protein and 1 uL buffer ) is better > >> than the other two cases.Right now it's growing in room temp > >> in about a week.And crystals that not fresh got some bubbles > >> around the edge and didn't diffract well. > >> > >> Does anyone have any suggestions that what I could do to > >> improve the diffraction? > >> > >> Thanks a lot. > >> > >> Jenny > >> > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
