Hi Savvas, If the very good suggestions you have already got from the ccp4bb do not help, try crystallization with agarose as an additive. Crystals form inside the very soft gel and they are hold in place by this meshwork. So, they are mechanically protected and do not fall down onto the bottom of the sitting-drop well. A final concentration of 0.1-0.2 % (w/v) agarose is sufficient. When you harvest a crystal cut generously around it with a microtool, pick it up (e.g. using a nylon loop) and do not mind if some agarose comes with it.
for example: Biertümpfel, C.; Basquin, J.; Suck, D. & Sauter, C. Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9 PMID: 12351881 Good luck! christian Savvas Savvides wrote: > Dear colleagues, > > we have been growing crystals of a protein complex in sitting-drop > geometry that stick to the bottom of the drop remarkably well. It’s as > if they are glued onto the plastic. This makes crystal handling next to > impossible without destroying the crystals. We have tried whiskers, > loops, all kinds of micro-tools, and pipetting techniques to no avail. > I can say at the outset that we have been unsuccessful in growing these > crystals in hanging-drops or at 4 degrees. Deglycosylating the complex > also leads to nowhere. In fact, we are only able to get crystals from > homogeneously glycosylated protein produced in HEK293S/I- cells. > > > > In the meantime we are playing with the idea of siliconizing the > sitting-drop depressions to alter the crystal/plate interface. But then > again, nucleation events on the plastic may be the reason we are > getting crystals in the first place. We have also thought of trying > microseeding to have more control on nucleation issues. Our protein > production is quite limiting and forces us to be very selective with our > experimentation. > > > > Nonetheless, while we are waiting for fresh material to explore some > of these ideas we would like to make the most out of the crystals we > have grown thus far. We would therefore very much appreciate any > input/ideas on manipulating these crystals for data collection. > > > > Best wishes > > Savvas > > > > > > ---- > Savvas Savvides > L-ProBE, Unit for Structural Biology > Ghent University > K.L. Ledeganckstraat 35 > 9000 Ghent, BELGIUM > office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 > Email: savvas.savvi...@ugent.be > http://www.lprobe.ugent.be/xray.html > > > > > > > > *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of > *Katarina Moravcevic > *Sent:* Tuesday, January 27, 2009 10:52 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] pseudo translation > > > > Hi all, > > here is a question from a beginner. I have a home source data set that > indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, > alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After > failing to get a MR solution with Phaser I ran the phenix.xtriage which > showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which > indicates pseudo translational symmetry. I was wondering if there is > anything I could do with this data to get around this problem. Given > that I don't have a lot of experience any suggestion/explanation would > be fantastic. > > Thanks in advance > > K > > > > * > > E-mail message checked by Spyware Doctor (6.0.0.386) > Database version: 5.11630 > http://www.pctools.com/spyware-doctor-antivirus/ > <http://www.pctools.com/en/spyware-doctor-antivirus/> > * _______________________________________________________________________ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA _______________________________________________________________________
