Pavel,
1.8.1-1168 also has this problem.
I found this problem existed with some dataset, but not all dataset.
1.8.1 give better map, so I built model with 1.8.1 map and go back to
1.7.3 before I deposit the structure to PDB.
When I compared the B factor from 1.8.x and 1.7.3, I found
B(1.7.3)=B(
Do you see the same MBP band (or corresponding to the same MW, in case it
isn't MBP) before cleavage, at the same total concentration of protein? If
not, your protein could be crashing out. Even so, if you use SDS and heat
up the sample, I am surprised that your protein just disappeared. TEV
protea
Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with a
(charged?) head. Partially occupied perhaps, as they're so close together.
On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar wrote:
> Dear All
>
> I am working on an enzyme which involved in fatty acid biosynthesis. We
>
We once had a more-or-less MBP-sized fragment before cleavage, but
this turned to be a spontaneous mutation. This expression experiment
had been started from a glycerol stock with an unknown number of
growth cycles prior to expression.
Starting from a fresh transformation with the purified an
I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage
as the existence of the MBP band post-cleavage would not have been noteworthy
if it had also been there pre-cleavage.
A time course or at least a time zero sample is always a good idea.
Truncation products prior to pr
@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
I think that is a wrong assumption.
He did not specify if he sees the MB
This looks like an output from SCALEPACK. Unfortunately, one has no way to
know from the output if 21 and 90 are strong intensities or not. One cannot go
by the I/sigmaI alone. For example, suppose there is thermal diffuse scatter
at these positions or perhaps there is a cosmic ray or radioac
Dear All
Thank you for all your replies, the buffer for the TEV protease that I have
used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I
have tried using this buffer without NaCl but the TEV protease precipitates
when dialyzing over night, as for using glutathione (r
Rana,
I understood that these proteases are very efficient in cleavage. One time, I
had a construct MBP-3C-protein, I never able to cleave this particular
construct while I could do well with other truncations.The MBP you are seeing
might be co-purified with DHBx and unless gel band intensity s
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear SDY,
if you can see extra density after MR into which you can even build or
correct the model it is a good sign your chose the correct space group.
Check the geometry of your model. I suppose it is very distorted - a
matrix weight of 0.03 sounds
Since you're seeing the MBP band, it sounds as if you're getting some cleavage.
If there were no cleave you would only see the fusion and TEV bands on the gel.
It may be that your protein is not stable/soluble without the MBP fusion.
Different buffer conditions may help if your protein is bein
You do not mention what buffer you are trying to do your cleavage in. You need
a reducing agent for TEV to work (e.g. reduced gluthionine, DTT,
mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease
and the presence of divalent metal ions will/eventually inhibit TEV. TEV
Dear CCP4
I am having a problem with cleaving my fusion protein and I would be
grateful if you advice me regarding this situation, I have an MBP-DHBx fusion
protein and I am trying to cleave it using TEV protease, I have tried different
ratios and different temperatures with different inc
Dear All,
I have few basic questions for which I need help. I have a
3.4 A data and I have processed it to P4.
1.
I used pointless to find SG, it suggests P41 21
2. But I see two strong intensities in systematic absences
Intensities of systematic absences
h k l
Intensity S
I also think Rpim, as commonly defined, is not directly useful for measuring
the anomalous signal.
Commenting on its use, though, I find it quite useful to judge whether one is
adding useful data
to already existing ones. In a multiple crystals context I always look at both
Rmeas and Rpim.
Sup
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