Suggestions so far have been good ones. However, the MiTeGen microtools kit:
http://mitegen.com/products/microtools/microtools_kit1.shtml
comes with a "MicroSaw", which is a 10-micron thick kapton saw that is
intended for this purpose. That is, you don't pry the crystal off the
surface, but rather rest this saw against the surface, bring it over to
the edge of the stuck crystal and then work it back and forth until you
have cut underneath the crystal. Did you try this tool?
-James Holton
MAD Scientist
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
*
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