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:
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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.
