Dear All,
We got crystals for a 35 KDa protein with 323aa including His tag and
linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M
Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals
with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG
Dear Jobi,
The usual suspects are:
- add another purification step. In fact I'd do that first:
* Ni-column
* remove His-tag
* Ni-column
* gel filtration
if you have done that: add another step to test for better crystals
- additive screens
- room temperature data set to check if
Dear Jobi,
For a crystal which is big in size(0.2-0.3mm is pretty big) while diffract
poorly, dehydration (increase concentration of the precipitant slowly) is a
good choice to improve diffraction, especially for those tends to crack during
cryo.
Also those regular optimization approaches:
Dear Jobi,
the paper of Heras and Martin 'Post-crystallization treatments for improving
diffraction quality of protein crystals' is really helpful.
Additionally, if you have lysines have you tried reductive methylation?
Good luck,
e
On Wed, April 13, 2011 12:34, Bingfa Sun wrote:
Dear Jobi,
Hi Jobi,
I also had some crystals that were highly sensitive to glycerol. In one case, I
found that DMSO at 10-15% can cryo protect it, also the crystal could grow in
the presence of 10% DMSO which essentially eliminated a soaking step. In
another case, I grew the crystals in the presence of
Bei,
How do you concentrate your media? We use tangential flow filtration.
If you get a good filter (Millipore Spiral wound TFF is one example)
it goes pretty quick, in ~2 hours you should be able to process
4L(concentrate+ dilute several time in buffer). We secrete proteins
from insect cells,
On Tue, 2011-04-12 at 22:54 -0400, Edward A. Berry wrote:
What about doing the Fourier summation at the precise location
requested,
in order to not calculate the map or interpolate at all?
Input would be the mtz file rather than map file.
eab
One advantage of map interpolation is speed -
Dear all,
Do anyone know the way to estimate the importance of mutation contributing to
the stability of protein?
Thanks for the help.
Heng-Keat
Hi Bei,
For the extracellular protein I worked on in graduate school, I
typically purified it from 4 L preps in LB media. The standard
protocol was to do a crude low cut with ammonium sulfate cut followed by
precipitation of the protein with a high cut. The pellet was then
resuspended,
Hi Keat,
Check this
http://cupsat.tu-bs.de/ (CUPSAT: Cologne University Protein Stability
Analysis Tool)
Hope it serves your purpose.
Gauri
On Wed, Apr 13, 2011 at 9:50 AM, Heng Keat Tam
t...@bio.chemie.uni-freiburg.de wrote:
Dear all,
Do anyone know the way to estimate the importance of
Hi Jobi-
You might want to try using drop ratios ---we have had great success with this
many times. My favorite additive screen is using the Hampton Research
Crystal Screen HT as an additive screen. I usually start by adding 5% to the
well---this has often yielded good crystals where the
Dear Jobi,
See if you can slowly increase the PEG concentration to 28 - 30% and that will
be a good cryo. Since BIS Tris Propane is the buffer, I think, 28% 3350 should
work fine.
If the crystals crack by going straight to 28% PEG 3350, 0.1M Bis Tris Propane
pH:6.5, 0.2M Potassium thiocyanate
Hi Heng-Keat,
you can try our new server (beta version) at:
http://babel.ucmp.umu.se/prosms/
Cheers,
/Uwe
On 2011-04-13 15:50, Heng Keat Tam wrote:
Dear all,
Do anyone know the way to estimate the importance of mutation contributing to
the stability of protein?
Thanks for the help.
Many thanks Jacob and Mark for your questions/suggestions. In response:
So what happened with the non-reducing gel? (If the DTT was fresh, there
should be no problem, but if not...)
The gel is very clear, it shows the same exact pattern as the reducing gels.
Both high and low MW fractions run
This notice is posted on behalf of Wim Hol. Please send inquiries to him at
the email address at the bottom of the page.
Postdoctoral Position Available
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Department of Biochemistry, School of Medicine
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Structural Biology of the type
This is much better than Coronation Steet!
http://blog.the-scientist.com/2011/04/11/multipole-wigglers/
Rex Palmer
Birkbeck College
Dear All
What is the best program to use for comparing two protein structures which are
very similar both structurally and wrt aa sequence? ie to get the rms
deviations both generally and in selected regions.
Rex Palmer
Birkbeck College
Hi Rex,
(not claiming it is the best) may be you can use structure comparison tool
that allows you to load a bunch of similar structures, superpose them and
corresponding maps, and it will highlight various differences (Ramachandran,
rotamers, secondary structure, etc..).
Have a look at page #17
Dear All,
I am wondering about the ranges of RMSD bond lengths and angles. What are
the acceptable ranges for these two values? Is there some statistics for
them?
Thanks and best wishes.
Hi,
I want to generate a mask using NCSMASK; however, whenever I tried to add
the SYMMETRY keyword, and output mask cannot be opened in coot. The
following is my script and I was testing on PDB# 2VZ8. Thanks in advance
for any suggestions:
ncsmask xyzin ${EOMDATA}/2VZ8.pdb mskout 2VZ8-ncs.msk
This has been discussed multiple times on bb, also BMC p640.
The restraint target standard uncertainty
provides an upper limit54 for a reasonable bond or angle r.m.s.d. from
targets
within the model (approximately 0.02 Å and 1.9deg, respectively), but makes
no
further assumption where these
Thank you.
On Wed, Apr 13, 2011 at 6:03 PM, Bernhard Rupp (Hofkristallrat a.D.)
hofkristall...@gmail.com wrote:
This has been discussed multiple times on bb, also BMC p640.
The restraint target standard uncertainty
provides an upper limit54 for a reasonable bond or angle r.m.s.d. from
On Apr 13, 2011, at 4:00 PM, Rex Palmer wrote:
What is the best program to use for comparing two protein structures
which are very similar both structurally and wrt aa sequence? ie to
get the rms deviations both generally and in selected regions.
Best is kind of subjective, but you can use
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