Dr. Stefano,
1) I would add FAD in all buffers after lysis,even after GF you could perform
dialysis with buffer supplemented with FAD.
2)for the second question I found this paper which could be helpful:
Large-scale preparation and reconstitution of apo-flavoproteins with special
reference to but
And include FAD at a few uM in your column buffers. Although if you are getting
two separate sharp peaks for protein and FAD, it doesn't sound like it is
bleeding
off during chromatography but rather already dissociated in the material you
apply
to the column. Take a spectrum of the material in
Hi Zbyszek
I think this has deviated significantly from twinning problems!
I certainly don't claim the 1998 study was typical. The crystal was large by
present day standards, no cryoprotectant was used and non uniform
drying/cooling rates might have occurred.
The Juers et. al. paper includes t
In cases like this, I keep FAD around during all steps starting from cell
lysis, purification, dialysis and even crystallization. In one case we added
FAD to protein such that the protein: FAD ratio was 1 prior to setting up
crystallization trials.
Hope that helps.
Harkewal
Sent from my iPad
Hi Stefano,
Wouldn't you be better off going the other way around, that is supplementing
your protein solution with FAD prior to setting up the crystallization trials?
It sounds as if the FAD is quite labile in your case, but it could well
stabilize the protein and hence give rise to better di
Dear All (those dealing with wetlab stuff..),
While purifying a FAD containing protein we lose part of the FAD (on the gel
filtration we clearly see two bands corresponding to holoprotein and free FAD).
We obtain crystals but diffracting to only about 4 A despite their beautiful
look. Our hypot
On 03/13/2014 10:55 AM, Keller, Jacob wrote:
Unless you are interested in finding curious objects, what would you do with
protein quasicrystal? The practices of macromolecular crystallography is about
determining 3-dimensional structure of objects being crystallized. Protein
quasicrystal are r
Dear collegues,
I'm working with a drug complexed protein structure that is having major
twinning issues. The drug has a single Br atom on a benzene ring, which I'd
like to use for orienting the drug in the binding site. I have various
anomalous data sets, ranging from 3.0A resolution, all sca
Hi Jacob
>Maybe we can get the author to repeat the study for the other usual-suspect
>protein crystals to find out the truth, but the score currently seems to be
>1-0 in favor of cell parameter shifts versus microcrystal orientation...
You don't mean me do you? The measurement you refer to was
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>Unless you are interested in finding curious objects, what would you do with
>protein quasicrystal? The practices of macromolecular crystallography is about
>determining 3-dimensional structure of objects being crystallized. Protein
>quasicrystal are really unlikely to diffract to high enough r
Dear Wolfram,
First of all you should make sure you have substantial twinning. Is the twin
fraction close to zero, then don't use a twin target. Is twinning detected by
several test, then there is a good chance your data are twinned.
As for validation, if you do not completely trust your R-facto
Dear colleagues,
this is a request for comments on the evaluation of crystal structures that
resulted from twin refinement.
>From http://www.ysbl.york.ac.uk/~garib/refmac/Tutorials/refmac_tutorial.pdf:
"Although Rfactors are substantially smaller with twin refinement than
without twin
refinement
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Dear Jacob,
Measurement of the reciprocal space maps at reflections with triple axis
diffractometry allows experimental separation of mosaicity and strain
(variation in unit cell parameter) effects. See eg Boggon et al 2000 Acta Cryst
D56, 868-880 http://dx.doi.org/10.1107/S090744495837 for
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