The hydroxyls were on the wrong carbons in the previous picture I sent. These are correct.JamesOn Jul 11, 2012, at 1:37 PM, Lukacs, Christine wrote:Hi all- I have a protein that crystallizes in I422, and diffracts well, between 1.3-1.7A. Beautiful density, slightly higher final R-factors than you
It looks like dityrosine, usually caused by radiation damage (I think UV is the usual culprit).http://www.nugowiki.org/images/thumb/c/ca/HMDB06045.png/220px-HMDB06045.pngJamesOn Jul 11, 2012, at 1:37 PM, Lukacs, Christine wrote:Hi all- I have a protein that crystallizes in I422, and diffracts well,
Dear All,
Thanks alot for your valuable suggestion.I hope I will find out the
solution now. As far as to giveup is out of question
Thanks once agin
Regards;
Bashir
On Wed, July 11, 2012 05:25, Tuhin Bhowmick wrote:
> Dear Muhammad,
>
> I had a similar case, and the crystals could indeed b
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Have you looked at a UV-vis scan, there are some characteristic bumps you
should see depending on the state of your FeS cluster, plus you can monitor if
oxidation occurs over time. If I remember it correctly 322 nm is where you want
to look at.
Jürgen
On Jul 11, 2012, at 6:22 AM, Jan Rashid Um
Hi Jan,
How is the Fe-S cluster deformed? What is the evidence? Has your colleague
done Fe and S analysis? There is a lot of missing information to this question
that prevents a much more thorough answer. Is the protein membrane associated?
Part of a larger complex? Besides the Fe-S cluste
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Thierry
> Hello all,
>
> Could someone please advise me on where to purchase Mercury Phenylglyoxal
> from?
>
> Many thanks,
> Vijay
>
>
> V
Jan,
I agree that the DTNB assay (Ellman's Reagent) can be used to quantify exposed
Cysteines. Alternatively, you could do site directed mutagenesis and mutate
significant amino acids responsible for the conformational change from the open
to the closed state, with hopes to lock the protein in
Ellman's assay may help you.
http://en.wikipedia.org/wiki/Ellman's_reagent
http://www.piercenet.com/instructions/2160311.pdf
Allan
Quoting Jan Rashid Umar :
Dear all,
I am working on a protein where I have to stabilize the closed conformation
of the protein using disulphide bond. The strateg
Jan,
If the Cys residues are accessible, you could try DTNB to quantify the
number of Cys, thus determining if they are reduced or bridged.
http://en.wikipedia.org/wiki/Ellman's_reagent
Kelly
***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Rae
Dear all,
I am working on a protein where I have to stabilize the closed conformation
of the protein using disulphide bond. The strategy to design the cysteine
mutants is based on the molecular dynamic simulations, and accordingly the
residues were chosen. The ultimate goal is to trap the ligand i
I *believe* that the ISC operon is utilized in E. coli for all FeS
clusters, regardless if Rieske or not.
I know that when deleted, activity of FeS containing proteins (succinate
dehydrogenase and Glutamate synthase) greatly decreases. See:
http://www.ncbi.nlm.nih.gov/pubmed/11432781
Thus it *shou
Okay, that settles it.
Thank you David and Pat for sharing the knowledge and wisdom.
Kind regards,
Vijay
On Jul 11, 2012, at 6:43 AM, Patrick Loll wrote:
I second the mythical conclusion. We also tried to make it and
failed 10-15 yrs ago (we came up with the di-iodo compound, if I
recall).
I second the mythical conclusion. We also tried to make it and failed 10-15 yrs
ago (we came up with the di-iodo compound, if I recall). Many of these
organomercurials are really tough to handle; I suspect that even if we had
succeeded in making the desired compound, it would have been as solubl
Good to know about this ISC operon!
Do you know if it is specific for Rieske-type His2/Cys2 Fe2S2 clusters,
or for FeS clusters in general?
Thus wild-type E. coli is already making three types of ISC clusters
for succinate dehydrogenase or fumarate reductase (although some help
might be needed fo
Urea?!?!? My arm hair went up.
Anyways, DLS (Dynamic Light Scattering) might help. Hard to imagine something
quicker and easier, provided that someone next to you already has a DLS machine.
(sorry for the late answer)
Carlos
Em 26/06/2012, às 15:10, Brad Bennett escreveu:
> Native PAGE (i.
Hi Vijay,
Not an answer to your question, but I tried and failed to source any
about 10 years ago. I had a chemist friend try to make some following
the a protocol we found on the web - which didn't work.
The consensus on the ccp4bb back then was that it was a bit of
mythical beast, and I never p
If you need reducing agent, the best choice is probably TCEP. If you
are to choose between the BME and DTT, I'd recommend DTT, since BME
tends to modify cysteines (sometimes in active site which may be quite
annoying, although post-crystallization DTT soaks can remove some of
these). On the o
Jan,
Do you express the protein with the *E. coli isc* iron-sulfur cluster synthetic
operon?
I found great success, see:
Daughtry, KD et. al. JACS 2012
http://pubs.acs.org/doi/full/10.1021/ja2111898
- Kelly Daughtry
***
Kelly Daughtry, Ph.D.
Po
Hello all,
Could someone please advise me on where to purchase Mercury
Phenylglyoxal from?
Many thanks,
Vijay
Vijay S. Reddy, Ph.D.
Associate Professor
Department of Molecular Biology, TPC6
The Scripps Research Institute,
10550 North Torrey Pines Road
Dear all,
two post-doctoral positions are available in Pavel Plevka's group at Ceitec in
the Czech Republic:
http://www.ceitec.eu/programs/structural-biology/x-ray-crystallography-ii/
The projects involve x-ray crystallography and cryo-electron microscopy of
human viruses.
If you are intereste
Hi Ian,
> > @Ian: You'd be surprised how well Refmac can flatten sulfates if you
> > have a chiral volume outlier (see Figure 1d in Acta Cryst. D68: 484-496
> (2012)).
> But this is only because the 'negative' volume sign was erroneously used
in
> the chiral restraint instead of 'both' (or bette
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On 11 July 2012 11:39, Robbie Joosten wrote:
> @Ian: You’d be surprised how well Refmac can flatten sulfates if you have a
> chiral volume outlier (see Figure 1d in Acta Cryst. D68: 484-496 (2012)).
But this is only because the 'negative' volume sign was erroneously
used in the chiral restraint
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In ccp4-6.3.0, the chiral volume of SO4 is marked as 'both'.
SO4 chir_01 S O1 O2 O3both
Tim
On 07/11/12 12:39, Robbie Joosten wrote:
> Hi Joel,
>
>
>
> I prefer the swapping of atom names, which is pretty much what the
Dear Amr,
Same here. I didn't put DTT or any reducing agent in my buffer. I got
crystals of this protein.
Allan
Quoting herman.schreu...@sanofi.com:
Dear Amr,
Every protein is different, so one cannot make a general remark.
However, in my experience disulfide bonds are normally not affect
Hi Joel,
I prefer the swapping of atom names, which is pretty much what the program
chiron does, over hacking the restraint file. The latter makes the problem
reappear as soon as you use your PDB file on a machine with an 'unhacked'
restraint file.
@Ian: You'd be surprised how well Refmac c
Dear colleagues,
When using the LINK command in refmac5 version 5.5.0109 between symmetry
related molecules as below
LINK PDC B 1191.61O3' DA B 125 1555 2555 p
LINKO3' DA B 1251.61 PDC B 119 1555 3555 p
LINK PDG C 209
Dear Amr,
Every protein is different, so one cannot make a general remark.
However, in my experience disulfide bonds are normally not affected by
mercaptoethanol or DTT. I have had one protein where the disulfide bonds
were not made, likely because the protein was produced in E.coli.
However, the
Dear All,
I will be grateful to your suggestions about Iron-Sulfur cluster protein
purification. My colleague has some problems with the purification and it
seems that the iron-sulfur cluster might be deformed, and protein is
aggregated. The purification is done under aerobic (normal condition), a
Well - the problem may well be here - in the REFMAC dictionary (see
$CLIBD/monomers.s.SO4.cif ) the chiral volume calculation uses at the order
of the O numbering around the S atom .
So if your O numbering is not right handed you will have the chiral volume
calculated as positive, not negative.
T
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Hash: SHA1
Dear Joel,
out of curiosity: what is "the Chiral volume outliers issue"?
Cheers,
Tim
On 07/11/12 01:00, Joel Tyndall wrote:
> Hi people,
>
> We are refining a structure with sulfates and we are getting the
> Chiral volume outliers issue. I understa
HI Joe
This and similar cases should be changed in the library from
_chem_comp_chir.volume_sign = 'negative' (or 'positive') to 'both'.
Then if you're concerned about the standardisation of the atom
labelling you can always swap the labels of 2 atoms in the PDB file,
but at least with 'both' non-s
Dear Eleanor
I (capital!) believe the original poster means
http://compbio.clemson.edu/delphi.php
Best wishes
Roberto
On 11 Jul 2012, at 09:54, Eleanor Dodson wrote:
On 11 Jul 2012, at 03:36, dengzq1987
don't understand the question really. What DELPHI do you mean?
Many programs output DEL
Could I make a request?
Could people who ask questions to the bb possibly lay out their questions
clearly, capitalize the word "I" and use the correct punctuation such as
question marks?
It makes it so much easier to read, and - this is more important - you
will get more helpful responses if you
On 11 Jul 2012, at 03:36, dengzq1987
don't understand the question really. What DELPHI do you mean?
Many programs output DELPHI (refmac, SigmaA etc ) and they are usually used as
input to a difference map using FFT
Eleanor
> Hi all,
> recently，I want to use DelPhi to calculate the Electros
Hallo every one,
i am working on one enzyme who has sulphur bridge , is using 2 mercaptoethnol
or DTT during handling this protein for crystallography is not desirable .
best regards
Amr
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