If you have good and bad crystals in the same drop, I've had success
pushing a crummy crystal into a good crystal and having it release that
way.
Additionally, once I realized this was going to be a long term problem, I
started coating the sitting drop depressions with a thin layer of vacuum
grease. The crystals just slid right off the grease and I never saw any
changes in the diffraction data to suggest the grease was giving me
issues.
Chris
On Wed, 28 Jan 2009, 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: [log in to unmask]
>>>http://www.lprobe.ugent.be/xray.html
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
>>>Katarina Moravcevic
>>>Sent: Tuesday, January 27, 2009 10:52 PM
>>>To: [log in to unmask]
>>>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/en/spyware-doctor-antivirus/
>>>
Christopher L. Colbert, Ph.D.
Instructor Phone: (214) 645 5944
University of Texas Southwestern Medical Center FAX: (214) 645 5945
6001 Forest Park Lane
Dallas, TX 75390
|