Yep,
mostly you should stay away from Tris as this is the worst buffer system when
playing with temperature changes. Tris for example has a ∆pKa/10˚C -0.31
Good, N.E. (1986) Biochemistry 5, 467
Jürgen
P.S. @Matthew, was this what you meant by "the Good buffers often not" ? or
just a coinciden
One of the other things you need to be concerned about with thermal melts
is the change in buffer pKa as temperature varies (I seem to remember this
being called the "beta" factor). Phosphate is used for CD melts regularly
because its pKa is fairly invariant with temperature. (A good reference is
Why not thermal denaturation in the presence of Sypro Orange and a realtime PCR
machine ?
Crowther et al. Use of thermal melt curves to assess the quality of enzyme
preparations. Anal Biochem (2010) vol. 399 (2) pp. 268-275
Or (shameless advertisement):
Hain et al. Structural characterization a
Hi Harsh
Something like sodium borate at pH 9.0 could be an alternative to phosphate
buffers. If you are looking at thermal unfolding above 220nm, then the choice
of buffer is less critical as many buffers and additives are problematic only
below 200nm.
If your samples require high salt conce
Sent from my iPad
On 20/03/2013, at 7:59 PM, Harsh Bansia wrote:
> Sorry for a simple and non-CCP4 question.
> I have determined the structures of three different mutants of a thermostable
> protein by X-ray crystallography method. I feel that Mg2+ has a role in
> protein stability.
> So I w
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Dear Kyriacos,
as David has pointed out different indexing possibilites might be a
problem. Could you run the mtz-files from mosflm through pointless
(and then aimless instead of scala)? pointless compares the different
indexing possibilities and pick
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Dear Zhijie,
this sounds like a graphics driver issue. You can use the command
'lspci | grep -i vga' to find out what graphics chip you have on the
computer, and you should check /var/log/Xorg.0.log what driver is
actually used. If it is an NVIDIA car
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Dear Herman,
the short answer might be that at the stage of COLSPOT the term
'resolution' has a limited meaning because COLSPOT does not rely on
the experimental setup like distance and beam direction, so the term
'resolution limit' is conceptually no
Hi George,
I think that part can be made from an aluminum or a steel tube with proper OD.
The ID seems only need to be large enough to allow the glass tube to fit in -
depending what is allowed by the two black end adaptors. You can check your
engineering department or some machine shops to fin
Hi George,
What is the plastic tube? Is it part of the end adaptors or it is part of the
tubing? What type of plastic it is? If you can provide a photograph of the
broken part maybe someone can give you more useful suggestions.
Zhijie
From: George Kontopidis
Sent: Wednesday, March 20, 2013
Hi Sebastino
Both PDB and PBDe have facebook pages. PDBe updates the page
weekly with some snippets as well.
On Wed, Mar 20, 2013 at 3:41 PM, Boaz Shaanan
wrote:
> Hi,
>
> I think that what you're looking for (or close to it) is available on
> the pdb site. Through your PDB login
Dear Colleagues,
Apologies for the non-crystallographic question but it very likely that someone
out there may have the answer.
I have a chromatography column. Unfortunately the company does not provide a
replacement for the plastic tube, which should not cost more than 100 €. The
cost for
On 03/20/13 13:25, Kyriacos Petratos wrote:
Dear All,
we have two data sets at about 0.9 and 1.9 Ang. resolution collected
from a single crystal.
Integration with iMosflm seems to be fine like the scaling within each
of the data sets.
When we try to merge and scale both of them with 'Scala' we
Dear All,
we have two data sets at about 0.9 and 1.9 Ang. resolution collected from a
single crystal.
Integration with iMosflm seems to be fine like the scaling within each of
the data sets.
When we try to merge and scale both of them with 'Scala' we get extremely
high scale factors for the lo
Dear Zbyszek,
I am concerned that the unmerged data would be bypassed and not preserved in
your recommendation. I also find it counter intuitive that the merged data
would then be unmerged into a lower symmetry and be better than the unmerged
data; there is I imagine some useful reference or two
I have found it is best to test the absorption of your buffer in the
wavelength range you are interested in.
If you are going to do a temperature study with CD perhaps at 222 nm, then
test your buffer there with your UV spec.
You want to have little or no absorption. Or do the range 200-270 nm and
Hi Boaz,
that is definitely what I was looking for.
Thank you very much.
Other suggestions, on weekly queries based on "your favourite protein" sequence
were given by Gerard Kleyvegt
(http://bip.weizmann.ac.il/noop/NOOP_seqalert.html) and David Briggs
(http://www.sbg.bio.ic.ac.uk/phyre2/html/h
Hi Zhijie,
our system administrator has warned me sternly against touching RHEL
6.4. The same might hold true for CentOS.
I would be interested to hear from anyone running 6.4 successfully.
Andreas
On 20/03/2013 2:42, Zhijie Li wrote:
Hello,
We have a curious situation here: after upgra
Hello,
We have a curious situation here: after upgrading CentOS 6.3 to 6.4, COOT runs
slowly every time after the "go to atom" command is executed - every rotation
of the molecules takes nearly 1 second to finish. But if the "go to atom"
command is never sent then the speed is normal. A COOT ru
Hi,
I think that what you're looking for (or close to it) is available on the pdb site. Through your PDB login you can do a search according to keyword(s) and ask to repeat the search periodically. You'll get an alert in the e-mail. I actually use it for
some time.
Cheers,
Formulatrix, Inc. located in Waltham, MA is seeking a senior product
manager for our protein crystallography software, Rock Maker. The candidate
will be responsible for prioritizing and defining features of Rock Maker as
well as working with customers to get their feedback. This person will be
invo
Tris-sulfate might be acceptable for studies in the presence of
magnesium ion. Na-MES is tolerable at low concentrations and shorter
path lengths if a cutoff of 190-200 nm or so is acceptable. Chloride is
indeed problematic in the far UV.
___
Roger S. Rowlet
Harsh,
This article describes common buffers for CD on page 2:
http://www.ncbi.nlm.nih.gov/pubmed/17406547
Or this article on page 8:
http://www.ncbi.nlm.nih.gov/pubmed/16027053
It seems like phosphate is the best, because it has low absorption at
180-200nm region. From organic buffers Tris/H2SO4
Hi Sebastiano,
Phyre Alarm would do something similar to what you suggest.
http://www.sbg.bio.ic.ac.uk/phyre2/html/help.cgi?id=help/phyrealarm
HTH,
Dave
David C. Briggs PhD
http://about.me/david_briggs
On 20 March 2013 12:40, Sebastiano Pasqualato
wrote:
>
> Hi a
Hi all,
just wondering if anybody is aware of a service similar to PubCrawler
(http://pubcrawler.gen.tcd.ie/), but for PDB structures, released and
unreleased.
Basically a weekly email that would alert you of freshly deposited structures
that match some keywords.
Thanks in advance,
ciao
Sebasti
However, these buffers generally absorb in the far UV (180-200nm) which is
the most interesting
part for discriminating the secondary structure distribution. Even if this
is not the primary interest, the 220nm
points one might want to use for a denaturation study can have quite some
background
cont
I would use a low affinity metal ion binding buffer like MOPS, HEPES, or
MES. The "Good" buffers all have fairly low metal-ion affinity.
Phosphates will be problematic because of magnesium phosphate formation.
Cheers,
___
Roger S. Rowlett
Gordon & Dorothy Kl
Dear Wei,
There is a CCP4 program called Convert to/modify/extend MTZ which you
can use to convert various file types (notably SHELX, CNS/XPLOR, mmCIF,
MULTAN and TNT) to the MTZ format. Look for it in the Reflection Data
Utilities module.
http://www.ccp4.ac.uk/dist/share/ccp4i/help/modules/
Dear Tim, but probably I should adres this to Kai Diederichs,
not including the resolution cutoff in COLSPOT and IDXREF is a feature of XDS I
do not understand at all. For most cases, it may not matter since only the
strong spots are used, but what are the advantages?
In fact there are disadvan
Dear steffi,
Can you give me a link? I did not find it in google.
Thank you!
Wei
在 2013-03-19 20:55:42，"Stefanie Becker" 写道：
>
>Am Dienstag, 19. März 2013 04:37 CET, Wei Feng schrieb:
> Hi!
>
>don't know if you already got the answer by now, but there is a small program
>called map2mtz t
Dear Edward,
I think you gave a very good summary of what might have happened in the
crystals. If the two domains diffract like a single crystal and interfere, F's
are added, if they diffract like two different crystals, I's are added. In
fact, one should model the "special" protein molecule in
Hello,
The way I do it is by manually editing the SPOT.XDS file (generated by
the COLSPOT step). Spots are arranged by order of decreasing intensity
in that file. So if you do down the file, select an appropriate
intensity cutoff and then remove all spots below that value, it will
have the ef
Sorry for a simple and non-CCP4 question.
I have determined the structures of three different mutants of a
thermostable protein by X-ray crystallography method. I feel that Mg2+ has
a role in protein stability.
So I want to perform a thermal denaturation study by CD spectroscopy both
in presenc
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