I second Chris's suggestions. These have worked well for me in the
past. You only need a very thin layer of the grease (i.e. keep
wiping until its almost completely gone) and it usually has no affect
on the crystallization.
Jeff
On Jan 27, 2009, at 3:51 PM, Christopher Colbert wrote:
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: 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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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
