Dear Guillermo,
I think the referee has a point because you are comparing two
different proteins to propose a model of 2 states even though the
enzymes are highly similar with conservation of key features. With 40%
id/60% similarity, even if core domain structure and length are
conserved, there ar
Dear all,
first of all sorry for this off-topic question. I am requesting your help to
find some papers to convince one referee about the comparison
of two different protein states.
In our manuscript we show the crystal structure of an
enzyme. This structure represents the enzyme after catalysi
Hi Sanjeev,
In addition to all the excellent suggestions (trying different buffers/salts,
co-expression, concentrator, incubate and set up crystallization screening,
checking SPR, fusion), I have a couple more suggestions:
1) related to your question about cross-linker: do you have experimental
I agree with Engin’s suggestions.
Our group crystallized a complex of mesotypsin with BPTI, where we measured an
inhibition constant (approximating Kd) of 14 micromolar, by mixing the two pure
proteins together in 1:1 stoichiometry. Actually we do that for a lot of our
complexes with higher af
for Linux you need the 3-pin mini Din connectors
On Tue, Jan 31, 2017 at 8:29 AM, Jun Dong wrote:
> I have made coot and pymol 3D work with NVIDIA Quadro K5200 under Windows
> 7 but I could not make it work under Centos 7. WinCoot was not able to load
> big virus maps, it crashed all the time. I
Hi Jun,
on CentOS 7 it works as described at
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Stereo#Nvidia_3D_Vision_2
, with a Quadro K620 and BenQ XL2420TX . Unfortunately this monitor type seems
difficult to find nowadays.
best,
Kay
On Tue, 31 Jan 2017 16:29:17 +, Jun Don
Adding to this: the current test set generation in freerflag has one brilliant
feature and that is reproducibility. If you misplace your test set, you can
reproduce the same one if you run freerflag on the same complete set of
reflections. This was an excellent design choice. It has proved very
Not observing a complex on a gel filtration run for a week complex
(micromolar) is not necessarily unexpected. It all depends on your
protein concentrations, the dissociation kinetics (remember, a gel
filtration run is not an equilibrium experiment - it dilutes and
separates your sample), etc.
If your goal is a crystal structure, this reference may be worth consulting.
The paper describes the use of centrifugal concentrators to make a complex of
two proteins that have Kd of 19 micromolar for crystallization. They mixed the
two proteins and then concentrated using a membrane that all
Hi Ville,
it actually does not affect the quality of the Rcomplete value that I am
computing, so from my point of you this discussion was rather a matter of
interest and you should only invest time in case someone else has a reason.
Best regards,
Tim
On Tuesday 31 January 2017 03:54:09 PM Vil
Dear Sanjeev,
You can try to clone both of your gene in pET duet vector to coexpress both
proteins in bacterial expression system and purify with Nickel affinity
exchange chromatography (both proteins will be his tagged). This will eliminate
the problem of critical mixing of both proteins in st
If your SPR data is correct, you'll need to use a micro molar protein
concentration (or higher due to dilution in the column) in your analytical size
exclusion chromatography assay. What concentration did you use? Also, ideally
the buffers in both assays should match. And maybe your SPR data is
You could also try co-expressing them, pull down the complex by his tag. Also
you can try equilibrating the SEC column in a low concetration of one of the
proteins, if you can express enough. It is a bit surprising that you saw no
peak for the complex though. Since you have SPR data, what are ko
We have had good luck with creating fusion proteins of the 2 proteins in
question (http://www.nature.com/articles/ncomms14076). If you don't know
how they interact, you would need to try different linker length and
different order of the 2 proteins in the fusion protein. It would also be
helpful to
I have made coot and pymol 3D work with NVIDIA Quadro K5200 under Windows 7 but
I could not make it work under Centos 7. WinCoot was not able to load big virus
maps, it crashed all the time. I really want to make coot 3D work under Centos
7. Has anybody succeeded at running coot 3D in Centos 7?
Dear all,
I am trying to stabilize a protein-protein complex. Our SPR study indicates
it is having micro molar dissociation constant. I tried to purify both the
molecule in complex form with size exclusion chromatography (mixed both the
protein in equal molar ratio and incubated at 4 degree for 1
Hi Tim
thanks for the clear explanation. Currently it separately and randomly
draws the flags for each reflection, but the reflections related by twin
or symmetry operations (NCS is ignored) get the same flag, unless the
'NOSYM' keyword is used. This doesn't guarantee that each flag occurs
the sa
Xiao,
I had a direct connection from display port to display port that had worked for
several years (Windows 7). Since last year I have been losing the stereo.
Often the control panel won’t show the 120 Hz like what you saw. Sometimes I
was able to forcefully add a 120 Hz resolution (using t
Hi Ville,
let's assume you have 5,000 reflections and want 50 reflections to be in the
same bin (i.e. flagged with the same Rfree flag).
You enumerate your reflections and create a parallel list with 50 zeros, 50
ones, 50 twos, ... 50 onehundreds.
Then you run through the second list, and swap
Hi Tim,
You could create a freerflag column of the right length outside CCP4 and then
just import it into CCP4. That would save you a lot of scripting inside CCP4
context.
Cheers,
Robbie
-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: Tuesday, January 31, 2017 03:
Hi Xiao,
did you try manually changing the Hz in the nvidia system control panel?
It is somewhere under Resolution-> adapt (or something like that, sorry, I
don't have an english version). There you can change the frequency.
If it doesn't work and you only see a black screen, the system switches
b
On Tue, Jan 31, 2017 at 03:29:15AM +0100, Tim Gruene wrote:
> I had long wondered how to flag the fields in game minesweeper with a
> deterministic algorithm. When I read about 'random sort and bin' I though
> this
> was quite a beautiful way. I wonder if there is any reason behind not doing
>
On Mon, Jan 30, 2017 at 09:02:33PM +0100, Tim Gruene wrote:
> when I ran freerflag with the keyword FREERFRAC 0.002, the number of
> reflection
> per flag (between 0 and 500) is very high, it varies between 17 and 1.
>
> Why is there such a high variation, instead of flagging always the same
>
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