I am currently trying to solve two datasets of two different protein-protein
complexes. One of the proteins is common to both complexes and there is a
deposited structure in the PDB (100% identical). The other proteins currently
have not been solved and share ~50% identity between them. Both com
Thanks for the all the comments on/off board. Especially for papers describing
this method as it may be handy if the structure is solved. The crystals are
reproducible. They have started to appear after 4 days. I can accelerate it to
a day using just 1.5M NaCl as a reservoir solution, though I g
Dear All,
I have produced crystals from a robotic screen (API) in two conditions.
However, I have not been able to reproduce them. They originally grew in 2.5
days (over the weekend, though it could have been sooner). I noticed that when
I first set up the tray, these two wells were clear but w
Thank you board for pointing out my error. I am now wiser and so is the PhD
student.
A PhD student asked me what causes diffraction anisotropy. Quoting from the
Diffraction Anisotropy Server webpage that it is caused by whole-body
anisotropic vibration of unit cells. He asked whether a colder cyrostream could
improve anisotropy. My answer would be yes, as colder temperatures wo
Is the phenix website down? Anyone know when it will be back up?
I got several comments on/off board for this. I have checked by Western
blotting if any expression was occurring. I did not see any expression in
either the inclusion body or soluble fraction.
It appears that I have solved the problem;
Several papers suggest that the 10-30 bases after the start
We have been expressing a family of human proteins as inclusion bodies in E.
coli, which we can be refolded and crystallized. However, three members show no
expression either as inclusion bodies or as soluble proteins.
The genes were ligated into the pE21d vector with either a His-tag or no
Hi
Hi,
I am trying to reindex a P212121 lattice to change the axis order from 32.62,
64.89, 103.16 to 64.89, 103.16, 32.62. Looking at the REINDEXING manual I can't
find how to reindex the lattice. Does anyone know how I should do this and an
example of the execute script that I should use?
Than
They have, it is call PDB_REDO. You can download the maps and refined structure
there.
http://www.cmbi.ru.nl/pdb_redo/
Dan
First of all, you don't say how long it took to first set up crystals, for them
to grow, harvest, freeze and collect data on. Secondly how long did leave the
peptide/substrate for your SDS PAGE experiment? If they are of a different time
scale e.g. 6 hours v.s. 30 days, it may be that your enzym
Hi all,
Just writing an update, as I have had two people contact me asking if I had
found a solution and if I could share. I would like to apologize first for not
phrasing my question to describe the situation simply. I had several
suggestions that Coot, and PISA could do it but actually can no
I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have
superimposed using Chain A. Several programs will produce the RMSD of Chain A2,
A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3,
B4... to Chain B1 when I have superimposed the structures relat
Dear all,
I have collected ~160 degrees of data on a new crystal form of a protein which
has already been solved. Data was processed with XDS and reindex, scaled and
truncated with Aimless. Both XDS and Pointless suggested a Laue group of
P6/mmm with a possible space group of P6122 or P6522. S
Bob Sweet has a powerpoint presentation but it took a long time to download the
file
(http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=27&ved=0CEoQFjAGOBQ&url=http%3A%2F%2Fcima.chem.usyd.edu.au%3A8080%2FMMSN%2Fevents%2FRAAW_Mar_2005%2FBob_Sweet_v2.ppt&ei=_jRxUqvjGeqrsASg8YDwBA&usg=AFQ
You may want to read:
Evolutionary conservation of codon optimality reveals hidden signatures of
cotranslational folding
Nature structural & molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 237
Here they suggest that the codon bias is such it allows translation to pause
and folding of the pol
Protein "A" must be co-expressed as it does not express well alone.
Have you tried separating A+B on the Nickel column by flowing 2M urea to help
"tickle" off protein B. The amount will vary depending on the affinity. In my
case (~50nM) requires about 1000-1700ml of 20mM Tris, 2M urea pH 7.5. Th
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
The following paper (which can be found at
www.wolfson.huji.ac.il/purification/PDF/Literature/Bondos2003.pdf
Detection and prevention of protein aggregation before, during, and after
purification. Sarah E. Bondos and Alicia Bicknell (2003) Analytical
Biochemistry, 316, 223-231
contains a table
I am assuming you are after something like a "difference" map of the two
surfaces.
http://www.pymolwiki.org/index.php/Map_set
Map_set command can average, copy, difference, maximum, minimum, sum and unique
Hope this is what you are after.
Dan
"Some people might squawk at 2MB attachments on a BB..."
Surely that is bark :)
I have been using the Duet system from Novagen (or whatever it is called these
days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins
did not work in either vector. Either, one protein expressed or the other. I
played around with the promotors (they are both T7) by changi
Dear All,
Thanks for all the replies on and off-board. I received around twenty replies
and the majority have spoken in favor of the QIAgen BSA-free anti-5His mAb from
QIAGEN. Not to be bias, a couple of people recommended the one from Abcam as
well.
Thanks again, Dan
Are there any good antibodies for His-tags on the market. I have never used one
and I heard several stories that they were poor and not worth the money, over
the past few years. The only post I could find on the BB was from 2008. Have
His-Antibodies improved or are they still rubbish.
Phusion requires that the primers are phosphorylated for mutagensis to work,
unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte
Dan
Isn't it true that we cannot even agree on what MAD stands for?
Is the following right?
M = Multiple-wavelength. I think everyone agrees to this, although I
believe I've seen the occasional (and sometime non-sensical) variant
A = Anomalous (I think everyone agrees, although this term should
reall
Why should we store images?
From most of the posts it seems to aid in software development. If that is the
case, there should be a Failed Protein Databank (FPDB) where people could
upload datasets which they cannot solve. This would aid software development
and allow someone else to have ago at
I may be missing something or someone could point out that I am wrong and why
as I am curious, but with a highly redundant dataset the difference between
refining the final model against the full dataset would be small based upon the
random selection of reflections for Rfree?
If you are unsure about whether the disulfides have formed treat a small amount
of protein with N-ethylmaleimide. If the disulfides have not formed, when you
perform mass spec on the protein you should see an increase of mass of 125Da
for each exposed cysteine.
A couple of people asked why the GST/His steps. The only way I can expressed
this protein is by co-expression. Co-protein is GST tagged and my protein of
interest is N-term His and C-term StrepTag. Even with the coexpression, there
is a major degradation product, the reason why for the strep tag
Does anyone know how many times roughly you can re-use a Streptactin column? I
know that contamination with biotin will destroy the column but the protein
that I am using has gone through both a GST and Nickel purification steps
before seeing the Streptactin column and I think lately that I have
There are least two types of negative results
1) Contradiction of previously published results. Negative results of this kind
is either they are wrong, you are wrong or it depends on the differences within
the experimental methods used. An example of the latter case would be SPR vs
ITC. SPR for
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