Dear all
There is at least 1 post doctoral position available at the Genome
Institute of Singapore in the lab of Dr Prasanna R Kolatkar. We are
working primarily with transcription factors related to stem cell and
developmental biology. Although the attached advertisement is primarily
for a
Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the presence of
0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
I intend to perform heavy metal derivatization. I read some literature which
suggested that I could carry out quick soak with 0.5M
Hi Amit,
http://www.doe-mbi.ucla.edu/~sawaya/tutorials/Phasing/references.html
And the (IMHO) seminal heavy atom derivative reference:
Petsko, G.A., Perparation of Isomorphous Heavy-Atom Derivatives
Methods in Enzymology, Volume 114, , pages 147-157.
should give you all the info you need.
The
2008/9/25 amit sharma [EMAIL PROTECTED]
Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the presence of
0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
I intend to perform heavy metal derivatization. I read some literature which
You should try collecting a data set at the Br- edge, and perhaps other
wavelengths for MAD. I would think that you will locate at least one or two
Br-'s, which should be plenty with only 9 kD. If you want, you could collect
similar crystals with either KCl or KI, then do either SIRAS or MIRAS.
Hi Amit,
Dr. Dauter has several excellent papers about solving crystal structures with
halide ions. Use this search string in PubMed (without the quotes) to retrieve them: dauter [AUTH] AND halide. The anomalous signal of iodide can be detected using a home x-ray source but the anomalous
Hello,
I had recently collected and solved the phases for a protein molecule using
CCP4 and the ShelXCDE SAD method in it. What I was wondering was that the
peaks for the three SE incorporated methionines are there as expected, but
there is one peak scored roughly as the second largest where
Unexpected peaks in a S-SAD experiment sometimes turn out to be chloride,
sulfate or a metal ion. I would suggest that you run shelxd with and
without the disulfide option (or with different numbers of disulfides)
to see which is best, and also run SHELXE with the -b flag set. This will
There is ALWAYS anomalous scattering. You do not have be at the
absorption edge to get it. The question is just whether your experiment
is good enough to detect it.
So your question of overlapping always has the answer Yes, but I would
remove the words absorption edge from your question.
Or you could try crystallizing the protein in the presence of KI or
NaI, and collect some in-house SAD data. You could also try to boost
the concentration and number of ordered halide sites by quick soaking
the crystals with a higher concentration of the iodide salt.
In my limited
On Thursday 25 September 2008 08:45:10 Michael Jackson wrote:
This data was collected at 0.97960 Angstroms which is close to the peak
Xray absorption edge for Se but does anyone know if a disulfide has any
absorption edge overlapping here?
Your case might be different, but it could also be a true Se signal from
Se-Cys incorporated into your protein during Se-Met expression. Depending
on the protocol you used, Se may get incorporated into Cys, especially if
only source of sulfur is Se-Met. We have seen such signals from Cys-
We have had really outstanding success using trimethyllead and
samarium acetate as 'quick soak' derivatives. Both are soluble to 100 mM
in most crystallization solutions and have large anomalous signals at the
copper-kalpha wavelength used at most home sources. A quick 30 sec to 5
minute
Dear all,
We have some troubles with the cristallization of glycosylated proteins and
want to try to deglycosate them. We have never done this so we want to know
what enzymes are the best (efficiency; less protein loss, ... ). And if it
gaves better results in crystallization screens.
Thank
Dear Eugenio,
I believe the paper by Chang VT et al 2007 (PMID: 17355862) might offer the
answers you're after (assuming N-glycosylation is your main concern). For
O-linked sugars, the situation is much more complex, reflecting their
diversity: mucin-type can usually be dealt with by construct
Hello Everyone
I've been trying to crystallize a zinc-containing enzyme for what
seems to me to be an eternity. The protein contains stoichiometric
zinc (1 zinc/ protein monomer) when isolated and the zinc is required
for activity. Each crystal we've obtained has lost the zinc and
Dear Crystallographers,
I remember having seen on this listserve that coomassie stain is horribly
non-linear in intensity per protein concentration, which leads me to two
questions:
1. Does anybody have a reference for quantitation of coomassie's linearity
(and possibly other stains), i.e.
Hi Eugenio,
It will depend on the expressing system you are using: mamalian
cells, insect cells, yeast.
For enzymatic deglycosylation I suggest you to do first do a small
scale test, using the following enzymes:
PNGase F is extremely efficient.
EndoH (you can try EndoHf which is cheaper and
Based on my own experience with zinc-metalloenzymes with thiolate
ligands, it's usually more a problem to get the zinc OUT than get it IN.
Zinc is pretty thiophilic, so removing it once ligated in a multiple Cys
environment is often difficult. Have you tried TCEP as a reducing agent
for
One thing to watch out for: in addition to frustrating crystallographers those
sugars play
other roles in protein chemistry, and its not uncommon for the solubility of
proteins to
change dramatically after deglycosylation.
Good luck!
MLM
We had a zinc-finger containing protein that we were soaking with different
heavy atom compounds. It turns out KAu(CN)2 provided the best diffraction
of several soaks. We found out it was because the gold had replaced the
zinc ion and was coordinated by the Cys and His's. The lab nickednamed
I don't know if anyone had experience of TCEP inducing zinc acetate to
precipitate. This paper mentioned this.
The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution
*
Paul C. Smith, Stuart Firestein and John F. Hunt
Journal of Molecular
Dear Sue,
This is a very interesting case! Normally the Zn-S bond is quite strong - so
it's an unusual situation to have. Did you already attempt to purify the
protein in the presence of tiny quantities of Zn? Also, what buffers and
other components are you using during purification, and what is
Please note that TCEP decomposes and one of the decomposition products is
phosphate. Enough TCEP and you might have Zinc Phosphate crystals which can
sometimes look very odd and protein looking.
Artem
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Jennifer
On 25 Sep 2008, at 14:35, Jacob Keller wrote:
Dear Crystallographers,
I remember having seen on this listserve that coomassie stain is
horribly non-linear in intensity per protein concentration, which
leads me to two questions:
1. Does anybody have a reference for quantitation of
Here is my two cents...
How strong zinc is captured by the protein is very protein dependent: I
always thought that a great case for this variability was the protection
of RING ubiquitin ligases against NEM. Cysteine-catalyzing HECT
ubiquitin ligase are killed by NEM; RING finger ligases,
I mentioned previously phenix.refine tosses your weak data if IMEAN, SIGIMEAN
are chosen during refinement.
I'm wondering if this omission of weak Fobs from the Fobs-Fcalc difference map
explains why the difference maps out of refmac seem to be more helpful in
showing where to move
The Bio-Rad Flamingo Fluorescent Gel Stain has a very linear profile
with protein concentration (if you believe the standard curves in the
manual). In my hands it gives nice results for binding assays.
Cheers,
-Greg Alushin
On Sep 25, 2008, at 2:35 PM, Jacob Keller wrote:
Dear
Jacob,
Coomassie fluorescence is linear over a wide range of concentrations
(citation below). You need access to an infrared scanner in order to
use this technique. I've used a Li-cor Odyssey scanner with good
results (http://www.licor.com/bio/odyssey/index.jsp).
Anal Biochem. 2006 Mar
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