Hi Akila,
In addition to what others have asked about the dialysis buffer, a few
more comments that might help to decide next steps because the
precipitation (note precipitation and aggregation are related but not
synonymous) may be due to several different or related reasons:
1) At what stage
Just to clarify, 1 and 2 is obsolete when using 3.
B
Hi All,
With Bernhard's help, I fixed this issue of render tool in Coot in Win7.
I summarize what I did here:
1.* - add C:\yourccp4installationdirectory\bin to PATH in the coot
startup script (runwincoot.bat in
Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an
old name for sulfuric acid.
> On Mar 29, 2017, at 8:40 PM, Keller, Jacob wrote:
>
>> And if we are going to pour scorn and vitriol on Tris, why not mention its
>> large dpKa/dT of 0.03 pH
>And if we are going to pour scorn and vitriol on Tris, why not mention its
>large dpKa/dT of 0.03 pH units/deg ?
Hah! That's what many people are doing when they make buffers: pouring vitriol
(HCl) on TRIS! I prefer to pour concentrated HEPES, and get two buffers without
adding any extra Cl-.
Just to point out that whatever buffer you purify your protein into is possibly
not the one that will keep your protein happiest. We had the opportunity of
testing about 250 proteins in DSF against 26 different buffer / salt
combinations (in triplicate, with lots of controls) and found out
Hi Everyone,
Does anyone know what’s the fastest way the find all commercially available
proline derivatives on carbon gamma?
Thanks,
Jan
> On Mar 29, 2017, at 4:15 PM, Chun Luo wrote:
>
> In addition to price, the prevalence of Ni purification may be another reason
> for Tris popularity. Some His-tagged constructs don't bind to Ni well in
> HEPES. I wonder if anyone has similar experience or comments. —Chun
In addition to price, the prevalence of Ni purification may be another reason
for Tris popularity. Some His-tagged constructs don't bind to Ni well in HEPES.
I wonder if anyone has similar experience or comments. --Chun
-Original Message-
From: CCP4 bulletin board
I heartily concur with Craig. Tris can be a dangerous buffer for many
reasons, including those listed below. In addition, as a primary amine,
it can complicate work with metalloproteins and has moderate
nucleophilicity. There is almost always a better buffer choice than Tris.
Best regards,
Mark
There are almost always better choices than Tris buffer.
Mo Cleland used to call it “Trash” buffer. He is no longer with us, but today
I will happily carry that flag in his honor.
Tris may show up in your crystal structure, especially at carbohydrate binding
sites.
Tris may be a surprisingly
Hi All,
With Bernhard's help, I fixed this issue of render tool in Coot in Win7. I
summarize what I did here:
1.* - add C:\yourccp4installationdirectory\bin to PATH in the coot startup
script (runwincoot.bat in C:\yourwincootinstallationdirectory) *
I use Notepad to open the .bat file and do
...it's just a wonderful tradition! there's an interesting description of the
history of tris in maniatis
cheers
jon
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von David
Briggs
Gesendet: Mittwoch, 29. März 2017 17:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb]
In addition to what Nicolas has pointed out, is it quite suspicious to me that you have the same multiplicity in the high resolution shell as in the low resolution. On Mar 29, 2017 18:17, Nicolas FOOS wrote:
Dear Juliana,
all the statistics
On 29/03/17 13:36, Adriana Sene wrote:
Hi
Hi,
I am new here, I am looking for some way to modify one of my ligand. I
have some cl atoms in the ligand which I wanted to remove and add
other functional groups. If some one can tell me, how it can be done
or which tools either free or paid can
If you have outliers - worry about why?
(By the way - what is the multiplicity?}
Look at the AIMLESS list of rejections/ the scale factor for different
batches/ reports of ice rings/ etc
There has to be a reason, and with snsible reprocessing you can probably
but much better resolution data
I agree that you should try to use all the data. There is nothing wrong with
solving your structure with the data you trust and then extending the
resolution when your model is in an advanced state of refinement. If you worry
whether your data has added value, you can use paired refinement to
Exactly. Tris is very cheap. HEPES not so much. On the other hand,
zwitterionic buffers have significant advantages in terms of controlling
inorganic anion or cation concentrations.
___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of
Dear Juliana,
all the statistics presented here looks good in terms of resolution cut
(maybe I will be less sever). For me the point is about the mosaicity
you report 1.90 it's high in my opinion. How looks you images? I am
wondering if the indexation is really right. And maybe the complain
One thing that sometimes helps me in this situation is reprocess and refine
in lower symmetry like P21. It could be you have pseudosymmetry and need
to model more molecules in the AU to better reflect your data. Sometimes
this can help. If that doesn't work, then you may have to stick with your
To be really convinced I think you should also compare the maps at 2.6 and 2.3
Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better, perhaps
you are adding noise, but the I/sigma and CC1/2 values suggest you aren’t.
Perhaps try 2.5 and 2.4 Å also.
And perhaps remove a
It doesn't cost as much as HEPES, iirc.
On Wed, 29 Mar 2017, 16:36 Keller, Jacob, wrote:
> A bit off topic, but I’ve always wondered how TRIS got so popular what
> with it’s pKa of 8.3—does anyone know?
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board
It is not clear to me why you believe that cutting the resolution of the data
would improve your model (which after all is the aim of refinement). At the
edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t seem to be
anything wrong with the Wilson plot. Th R-factor will of
Probably the price, HEPES is nearly 5-8 fold more expensive. PBS is fine,
unless you have to add divalents. TRIS is your (cheap) friend!
Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel:
A bit off topic, but I’ve always wondered how TRIS got so popular what with
it’s pKa of 8.3—does anyone know?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger
Rowlett
Sent: Wednesday, March 29, 2017 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein
Hi Adriana,
I support Johannes answer, but personally, I prefer to write directly
the smiles string for your ligand and then modify it.
Since the code depends on the starting atom, it could be tricky to
determine where to mutate if you did not write it by yourself in first
place.
You can
What are you dialyzing against? Your storage solution should typically
be buffered away from the pI and contain at least a small amount of
kosmotropic salt, e.g. NaCl. Some proteins will require additional
stabilizing/solubilizing agents such as glycerol or reducing agents.
FYI, Tris-Cl, pH
And what are you dialyzing it against?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Akilandeswari Gopalan
Sent: Wednesday, March 29, 2017 9:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein precipitation reg
Dear all,
I am a PhD student doing structural
Hello,
I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and CC1/2 =
0.779, the summary data is below), but when I perform Xtriage analysis it says
that “There are a large number of outliers in the data”. The space group is
P212121. When I refine the MR solution the Rfree stops
Dear all,
I am a PhD student doing structural studies on a few proteins from
Mycobacterium tuberculosis. The gene encoding the proteins I work on are
cloned into pet22b with c terminal His tag. the proteins are expressing
well. upon purification I am getting good yield of protein but during
Anybody notice that the CCP4 wiki is down?
Dear Colleagues,
I'm happy to promote an opportunity for a postdoctoral associate in my lab. Our
principle interest is the structural and function of vitamin D related
cytochromes P450.
We also interface regularly with the state of the art Hauptman-Woodward Medical
Research Institute. More
Hi
I am new here, I am looking for some way to modify one of my ligand. I have
some cl atoms in the ligand which I wanted to remove and add other
functional groups. If some one can tell me, how it can be done or which
tools either free or paid can be used to do that.
bet
Adiana
Dear Colleagues,
We draw your attention to the next IUCr Diffraction Data Deposition Working
Group (DDDWG) Workshop which is entitled "Research Data Management" to be
held at the ACA 2017 in New Orleans.
Details can be found here:-
http://forums.iucr.org/viewtopic.php?f=21=395
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