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: CCP4BB@JISCMAIL.AC.UK > 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 >
