Dear Mengxiao,
From my experience I would say that the two e. densities (blob1 and blob2)
are present the same molecule.
What that might be is more difficult to answer.
Based in the concentration you gave us and the electron density volume I
would say that is more likely to be ammonium sulphate
I think that SeMet oxidation has been a problem in the past in at least one
case that I know, that of TolC by Koronakis et al. The same group addressed
these problems in more detail in a second paper (see below):
Crystal structure of the bacterial membrane protein TolC central to
multidrug eff
This post has a question and an answer (good karma)...answer first
In response to Matt's question about periplasmic harvesting, we do an
osmotic lysis of e.coli by a series of centrifugations. First, harvest your
cells normally (4k 15 min). Then vigorously resuspend in a 20% (w/v)
sucrose, 1 mM ED
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4C8PFBS-2&_us
er=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C50221&_version=1&
_urlVersion=0&_userid=10&md5=aee9aceae8dfa17b363ee2ec634debb0
Osmotic shock is OK, or you can try chlorophorm disruption. You can Google
for eit
Hi Matt,
Why not check the NEB pMal system manual
(http://www.neb.com/nebecomm/ManualFiles/manualE8000.pdf). It has a good
cold osmotic shock protocol that I've been using for my periplasmic MBP
fusions.
Zhijie
- Original Message -
From: "Matt Colins"
To:
Sent: Monday, February
Hello all,
We've recently installed the newest version of PHASER, and when I do a
brute rotation function, I don't get an .rlist file output even though the
job completes successfully. Has anyone else had this problem, and is
there a way to force PHASER to output an .rlist? (I haven't been able
Hi,I believe it is not important as long as you run a proper scan of the
crystal. Both forms will allow proper phasing.
This is very well described in a paper from Thomazeau et al.
here is the reference
MAD on threonine synthase: the phasing power of oxidized selenomethionine.
Acta Crystallogr D B
Hi,
I wouldn't bother either. I once phased a structure based on about 160
oxidized Se in the AU (Hothorn et al., JBC, 2006). Just make sure that
most of them are oxidized and do a proper absorption scan.
best
Michael
aka akaka wrote:
Dear All
I would like to know whether oxidation of Se
Hi,
I wouldn't worry about Se oxidation. In principle having a mix of
oxidized/reduced seleniums is unfavorable, as you'll have less signal at the
edge (broadening). However, all-oxidized Se apparently makes things better
(sharper and more intense peak; I forgot the reference, i think it may
Dear AllI would like to know whether oxidation of Se entails any problem for
SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing
agents in my protein (extracellular and disulphide bonds are
important).ThanksDr. R.DepetrisWeill Cornell Medical College
___
Applications are invited for a postdoctoral position to work on the
structure determination of rhomboid intramembrane proteases critical
in cell signaling pathways. These proteases have been linked to human
disorders such as hereditary blindness and breast cancer. Our aim is
to understand t
On 10:43 Mon 09 Feb , Jacob Keller wrote:
> can anyone recommend a nice, free software package for analyzing
> binding curves? The application I have in mind is fluorescence
> polarization studies between a FITC-peptide and a protein, for which I
> already seem to have good data.
Try fityk
Dear all,
I plan to prepare a protein fused to pelB signal peptide and secreted to
bacterial periplasmic space. Does anyone have a detailed protocol
for harvesting the protein from bacterial periplasmic space? The protein needs
to be in the native state and will be purified on a nickel column.
Hi Ed,
A bit late on the subject
I have collected atomic resolution data (around 0.97A) on both bovine
and porcine phospholipase A2 crystals which at
the time of data collection were between 10-16 years old.
Crystallization setup was liquid-liquid diffusion in glass capillaries.
Diffe
I have used qtiplot to fit nmr titration curves.
http://soft.proindependent.com/qtiplot.html
Another open-source app for data analysis and plotting
http://scigraphica.sourceforge.net/index.html
I run ubuntu linux and they are both available in the ubuntu repositories.
Luke Kontogiannis
*
The folder unfortunately is gone. I played with this more and it seems that
not only does the TLS Create/Edit module have this error, the ccp4mg module
does as well. If I "abort" the process to remove the directory the project
file and contents then are removed regardless of input but the fold
Hello All,
can anyone recommend a nice, free software package for analyzing binding
curves? The application I have in mind is fluorescence polarization studies
between a FITC-peptide and a protein, for which I already seem to have good
data.
Thanks in advance,
Jacob Keller
**
Hello Marek,
182 kDa protein is nothing special - unless it has huge areas of disorder,
membrane-association domains, coiled coils, or something like that. Much
larger proteins and protein complexes have been successfully crystallized.
With respect to purification - this is where you may want to
Hi Andy,
where shall I start?
1.) Where is your probe.exe and reduce.exe? These should either be in
your global PATH or in driveletter:\YourWinCootDirectory\bin.
2.) if you want to point to specific probe/reduce.exe files. These
should be given in the .(dot)coot.py file (which in WinCoot is rea
Hi Marek,
I agree with Tim. 182kDa is certainly not too large for crystallization.
As for purification .
Have you tried different buffer conditions for your Ni-NTA purification ?
Adjusting the pH or salt component may improve the purification sufficiently.
You could try changing the salt c
Dear all,
I am trying to use the molprobity server with WinCoot 0.6-pre-1.
I have followed the instructions here
http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.html
and believe I have put probe and reduce into the right places and
pointed to them in the coot.py script.
however, when I lo
Hi,
182kDa is large I'd say, but not too large for crystallisation.
Since you are using a Ni-column anyway you could re-clone it with a
His-tag that is cleavable by an enzyme (TeV, FactorX,... - I am a little
out of date about which enzyme is the most favoured on nowadays).
This would add on
Dear all,
I´m new in the field of crystallography. So please
excuse my first question is off topic. I´m trying to purify a 182 kDa
his-tagged bacterial enzyme. The expression in E. coli works quite
well. After Ni-NTA the protein isn´t pure enough at all, so I tried
several gelfiltration columns, b
What are you assigning as FP and FPH?
Can you send the complete command script?
You have set a "real occupacy to 1.0 which is not appropriate if FP and
FPH are equal - it should be 0.000
But I await you complete script..
Eleanor
Alpharyun Ni wrote:
> Hi everyone!
>
> I have a problem when I use
Xie Jiabao wrote:
Dear all,
I am using the density modification tool in ccp4 to generate improved phases
for/from my model. I find that the electron density map I generate using Fobs,
and density modified phases (PHIDM) are not the same as that generated using
Fobs, phicalc (original calculat
Points to think about.
1) Do your data statistics indicate twinning - if they dont iyt is mst
unlikely to be present
Look at output of truncate ( new Ctruncate is better)
Get phenix xtriage report..
2) SAD phasing - I presume you knw it is P41212 and not P43212?
3) Going to a lower symmetry.
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