Hi Sandy,
If your protein is soluble (i.e. we're not dealing with brown
precipitate), it is possible that the brown color is due to a bound
iron-sulfur cluster. In this case, your protein was always brown, but
you couldn't see the color when it was very dilute. As a quick test,
you could
You've gotten some helpful replies already. I have found the following
reference to be helpful in understanding some of the physics behind
damage incurred during the crystal cooling process and a general
strategy to help avoid it. It expands upon what's already been said -
that larger
Umar,
Check out: Czepas et al. The impact of Lys--Arg surface mutations on
the crystallization of of the globular domain of RhoGDI, Acta D (2004)
60 275-280. They point out that sulfate ions can help mediate contacts
between arginine residues from neighboring molecules in the crystal.
To the CCP4 community.
As always, I have received a number of very helpful suggestions to my
query. I am grateful to all those who replied. My best summary of the
suggestions is below.
Best Regards,
-Andy Torelli
A notable consideration (provided by Jim Pflugrath) is that A JPEG has
someone must have done this, but I haven't been able to find one.
Thanks,
Andy Torelli
James,
I think the standard suggestions apply. Try to tweak your
crystallization conditions to get a single crystal (i.e. not a series of
stacked plates). PEG 400 can be a good substitute for MPD, but you
could also try additive screens as well as co-crystallization with
products,
Ru Heng,
It is commonly helpful to combine your protein and DNA under
dilute conditions and then concentrate the complex. Combining
concentrated DNA and protein together has a very good chance of
precipitating in my experience. I completely agree that trying
different buffer
Mark,
I agree with Artem in that you may find significant differences in the
purity of eluants from the 'high fidelity' IMAC resins (I use pre-packed
HisTrap columns).
You may also want to have a look at the following reference for a
comparison of a variety of different affinity tags in
Hi Kien,
You may also want to try to wash your column with elution buffer
(without reducing agents), equilibrate it with binding buffer (plus
reducing agents) and then bind your protein.
In the case of HisTrap columns (a pre-packed, Ni(II) resin column),
this procedure is recommended if
able to get these commands to work and I haven't found a
fix from the documentation, CCP4wiki or Google searches.
I would be grateful to anyone with an example script using these
commands they could share. If so, please spare the bulletin board and
respond to me directly.
Best Regards,
-Andy
native graphics software/hardware.
- You have to reboot the machine to change the memory configuration,
but people agreed the software is generally easy to configure for
resource allocation.
Thanks again to all who replied,
-Andy Torelli
--
=
Andrew T
running refinement within the guest Linux? My Google and
CCP4bb searches have not turned up anything so far.
Thanks in advance for any advice or reference material. I will post a
summary e-mail as well.
Best Regards,
-Andy Torelli
--
=
Andrew T
Hi Liew,
There have already been some very good suggestions. I agree with Tim
Gruene that a great starting point is to test the diffraction properties
of your crystal at room temperature. This can serve as a baseline for
comparing/evaluating cryoprotecting agents and methods.
You can
crystallography and the documentation I've
looked at for popular refinement programs that offer both targets do not
provide guidelines as far as I can tell. If anyone could recommend some
literature, I would really appreciate it.
Thank you very much for your time,
Best Regards,
-Andy Torelli
14 matches
Mail list logo
