Dear CCP4 board
I have a protein that exists in equilibrium between monomer and dimer and I'm
trying to calculate KD using size exclusion. The problem is that the column
dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5
uM in dimer fraction and 2 uM in monomer
Hi Careina,
I'm not sure you can assume that the ratio of monomer and dimer will stay
constant through the column - as you say, the protein is diluted during the
run, the ratio will change, unless you have a super tight dimer - which
clearly you do not. Also, as the mass and the molar extinction
Seems it's worth thinking about this as an experiment that has actually
been done: BB and wiki have been available in parallel for many years
now; so where has all the activity happened, where do people go for
information - and more to the point, where are other people happy to
volunteer
Hi Raji,
i the kit i used for this purpose was from Pierce it's call Protein BCA RAC
assay. BCA : for the colorimetric parts, en ad the RAC is for Reducing agent
compatibility. This RAC is also efficient with detergent. (according my
remember)
But you if you use at the same time detergent
The problem is that if you put detergent or reducing agent in Bradford or BCA,
the reaction is complet also without protein. You can't determine the color
gradient because every tubes are blue or purple.
De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK]
I agree with Dave, and i suggest one more method to estimate Kd,
The intrinsic fluorescence of proteins thanks to the aromatic chain side.
Maybe it's also possible to have an estimation with native gels if you use prot
A concentration as fixed and B protein concentration as variable. I am not
Dear Carina,
An additional problem is that due to the dilution, but also due to the
separation of monomers and dimers you get a reequilibration which is dependent
on Kon/Koff of the interaction. Unless these are very slow, you cannot use size
exclusion to determine the monomer/dimer ratio.
Hello Ian and ccp4 community,
I think you must use modified residue, N-acetylmethionine with code AME,
instead of LINKR.
May be someone find this mildly useful:
there is a file called mon_lib_list.cif, located in
$CCP4/lib/data/monomers/list/
If you are not sure about particular residue
I think that more people read wikis than contribute to them, so this
'experiment' (see below) is
a little biased. On the other hand, it is often easier to put a question
to CCP4bb than to search
for the answer in wikis and other documentation, however well organized
they are.
Kay: can you see
I couldn't help noticing the use of the passive voice in the last
sentence... :) Which is the real problem: wikis are an SEP, a
Somebody-Else's-Problem. Emails are not.
(And this interesting meta-conversation would never have happened on a
wiki.)
On 14/02/2014 09:15, George Sheldrick
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Dear Faisal,
I find the files in the directory $CLIB/data/monomers/list very useful
for such questions, e.g.
# grep -i ribulose *
mon_lib_list.cif:5RP 5RP 'RIBULOSE-5-PHOSPHATE non-polymer
mon_lib_list.cif:HMS HMS
Dear Sun,
I had a similar problem. If you have a good TaBr dataset this should
give you good phases.
You can combine the TaBr phases and Molrepl phases in SHARP.
What worked in my hands very well and is easy to do: SAD using Phaser
and the partial Mol Repl Model.
You find here a input file
Thanks everybody for sending me the three letter code..
regards
Faisal
On 2/14/14, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
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Dear Faisal,
I find the files in the directory $CLIB/data/monomers/list very useful
for such questions, e.g.
#
Dear George,
Ever since I read your message that gave rise to this thread I have had
a few remarks in mind that other preoccupations have kept me from sharing,
but perhaps there is still room for them among the numerous contributions to
this topic that have been made since.
What
The very best advice is: get better data if at all possible. Things
are much easier and more informative at 3A than at 4.2A and even
better at 2.5A.
There are various tricks which might help improve diffraction..
But if you are stuck then:
The partial MR phases are very useful for checking your
Frank,
the way you phrase implies that the outcome of the 'experiment', as you call
it, is obvious. But that would be throwing out the baby with the bathtub, it
seems to me.
My point is: the wiki and the bulletin board have complementary roles, and it
makes sense to use both.
After all, there
Hi Evgeny
Thanks a lot for responding. It's a nice idea but sadly it doesn't work.
For sure it makes the acetyl group planar at what was the A0-A1 link (now a
single AME residue), but the amide group at the A1-A2 link is now
pyramidal! Presumably now planar link restraints for this link are
In the past I remember using a simple program or webserver to check whether
modelled water molecules might be metal ions, especially sodium, magnesium etc.
as they do not differ much in density to waters. Based on coordination.
However, I can't remember the name and can't find it in google or
Hi Ian
dod you try without link and standard ACE atom naming. Refmac should be able to
deal N-terminal activation and few other things. At least it was the intention
when it was written. Bugs may have been (self)introduced to prevent this from
happening. If it is so then I would like to know.
turns out some people indeed have better memory than me:
WASP inside STAN server
http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl
thanks!
On 14 Feb 2014, at 12:49, Mark van Raaij wrote:
In the past I remember using a simple program or webserver to check whether
modelled water molecules
Gerard's beautifully worded message underscores in my mind what a great
resource for all kinds of information this bulletin board is. I have many times
been impressed by the time people take to answer queries, even if they
sometimes seem perfectly googlable to me.
As a community we can be
Sedimentation equilibrium or sedimentation velocity experiments by analytical
centrifugation is the best method for this.
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas
[nicolas.f...@synchrotron-soleil.fr]
Sent: Friday,
On 14/02/14 09:15, George Sheldrick wrote:
Perhaps someone should make a list of the questions most frequently
asked on CCP4bb and
the most helpful replies that they generated. This could even be made
into a wiki.
RIP minor-groove :-(
Hi,
I am a long time user of XDS (20 years this year) but all the same I find that
I have constant angst about losing observations because I don't understand what
goes in in the conversion steps to get to CCP4 format. I used to believe that
XSCALE was always necessary, and I always use it in
No, because Bradford is based on the increase in absorbance when the dye moves
from a hydrophilic environment to a hydrophobic one (like the protein interior,
or like the interior of a micelle). When detergents are present in excess of
their CMC, the change in absorbance from partitioning into
Dear CCP4 Users,
An update for the CCP4-6.4.0 series has just been released, consisting of the
following changes:
* phaser
* update to 2.5.6
* ccp4i
* phaser_EP: corrected HySS output parsing
Note that auto-updates work only with CCP4 6.4.0 series, therefore please
upgrade
I am trying to find out where I can get the fluorescent material (just a small
flat piece) I can glue to the tip of a pin for aligning the X-ray beam of our
home source. Does anyone know?
Thanks in advance!
Ronnie
Hi Careina,
Since alternative methods are being suggested...
ITC can be good for quantitating a monomer-dimer equilibrium by
diluting dimers out from a concentrated solution (which obviously
favours the dimer) - and presuming a reasonable Kon/Koff.
Alan Cooper has kindly figured out the data
Hi Ronnie,
We use YAG discs (Yttrium Aluminum Garnet). We buy small 10mm x 100um or 500um
thick discs, break them into shards, glue them to various alignment jigs and
they provide a very effective X-ray visualization tool.
Our latest supplier is Star Tech Instruments
Hi Tim,
I would actually recommend using pointless (or xprep) instead of
xdsconv. It is much easier to use and maybe even less error prone.
I take the point about pointless but XPREP is commercial software sold by
Bruker and costs €700 (as I remember), so is not really an option for everyone.
What a nice idea this ITC dilution is--a great example of a wet lab technique
learned en passant on the ccp4bb.
I wonder what range of Kds could feasibly be measured with existing calorimeter
sensitivities?
JPK
-Original Message-
From: CCP4 bulletin board
Hi Garib
I think that was the first thing we tried but it gave very poor geometry
for the ACE and also it's giving a VDW outlier for the ACE C - MET N bond.
So it looks like it's not recognising the link.
Cheers
-- Ian
On 14 February 2014 11:51, Garib Murshudov ga...@mrc-lmb.cam.ac.uk wrote:
Limited contributions are a common problem with wikis. This paper describes a
way to add some incentive to contributing to a wiki:
http://bioinformatics.oxfordjournals.org/content/29/14/1837.full
Cheers,
Robbie
Sent from my Windows Phone
Van: Kay Diederichs
That's an extremely useful link - thanks to Will Stanley for posting
that one.
For a VP-ITC machine I'd guess that you need to load the injector with
about 500ul of protein at a concentration of 80x the Kd or more.
Notice that Alan Cooper was injecting 10 microliters of protein at 2mM
with
Hi Robbie,
I think lack of feedback is a serious problem for wikis. I once started
an XDS tutorial at Kay's XDS-wiki, stating that it was still under
development and encouraging readers to send me an email if they want to
see faster progress in the completion of the tutorial. I never received
a
It is an interesting observation, Tim.
The fact that people do not read tutorials or manuals, is somewhat related to
the fact
that many questions asked in the ccp4bb could be answered by a more experienced
colleague,
a supervisor, a manual, a wiki, St Google the revealer, not to mention the
We've used CdWO4 crystals for years. They are highly radiation resistant and
colorless and give VERY bright fluorescence. Saint-Gobain has them in various
forms.
In principle you can mix your own CdWO4 from CdNO3 and NaWO4 ... under proper
conditions you let the solution digest to form
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