Dear All,
Sorry for the non crystallography related question
We are performing a fluorescence based assay to screen for inhibitor
compounds of our enzyme and ultimately crystallize the enzyme along with
inhibitor.
We see that some of our compounds are autofluorescent and thus are effecting
Charles Ballard schrieb:
Hi Francois
configure linux_intel_compilers
should do the trick. We distribute ccp4 built with the intel compilers for
OS X. Part of this is that the macs cover a much smaller range of cpus than
linux boxes, so optimisation is less of a problem. If you want
On 19/05/10 09:40, Kay Diederichs wrote:
Charles Ballard schrieb:
Hi Francois
configure linux_intel_compilers
should do the trick. We distribute ccp4 built with the intel compilers for
OS X. Part of this is that the macs cover a much smaller range of cpus than
linux boxes, so
Hi ccp4bbers,
I want to know if anyone has any experience about seeding (streak seeding or
microseeding) in microbatch under oil crystallization. I wonder if the oil
might block or wipe away the seeds if cat whisker is used for streak
seeding. Thank you for your input ahead.
Best regards,
Hi Jay,
there have been a few discussions about sigma/rms levels, absolute
values (e/A*3), significance levels etc on ccp4bb. See e.g.
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg06969.html
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg15273.html
which are the ones I could
Hello Donghui
we used a robot to setup the batch crystallization screens with some
microseed stock solution and the relevant protein and reservoir
stocks. That worked fine. I only mention a collegues work here, so if
there are more detailed questions, I will approach him.
Good luck,
Harm
On Wed,
On 19 May 2010, at 00:36, Paul Emsley wrote:
Jay Pan wrote:
Hello Everyone,
I have a reasonably well fitted electron density map through molecular
replacement. However, there is always some red region left no matter how
hard I tried when the mtz file is loaded into Coot. Is this because
Not quite correct, look into Blue Native PAGE. There you can seperate
natively by mass.
Jürgen
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street,
Dear Patrick,
I have Oryx robot in our lab. I got my initial crystallization hit from
sitting drop vapor diffusion method. This hit can be reproduced by sitting
drop through spontaneous nucleation with many tiny crystals after around 1
week but seeding into sitting drop failed and I found once
Hi Donghui,
I tried the microseeding using Hampton seeding tool
(http://hamptonresearch.com/product_detail.aspx?cid=18sid=157pid=448) for
under oil microbatch (without caring about wiping out seed by oil). It is
possible to seed the microbatch drop this way.
cheers, ravi
Ravindra D. Makde
Interestingly, Maxwell's demon pops up here, wh... ,
don't do it.
If you change the reaction rate in one direction 1000 times slower
than
in the other direction, then the reaction becomes practically
irreversible. And the system might not be at equilibrium.
Maia
R. M. Garavito
You are measuring fluorescence changes which are likely to be due to
compound binding by your enzyme.
In this case your blank must be your compound blank and no other.
Then you measure changes in fluorescence intensity and lambda max of
emission as you add your enzyme.
I do not know you
Maia speaks about native PAGE for which protein mobility (migration)
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a 2D
gel: Native in the first direction, then SDS-PAGE in the second one.
You
Dear Jacob,
I know that this is not the answer you were seeking, but for a modest
increase in the amount of protein required, a couple of analytical
ultracentrifugation experiments would be able to determine
stoichiometry and binding affinity for such a system. AUC has the
added benefit of being
You don't necessarily need the second dimension.
BN-PAGE gel:
protein A alone| some proteins you know the size as reference|protein B alone|
your mixture
You will be able to see in the mixture a) one or b) multiple bands, since the
Coomassie is equally distributed and attached to your protein
Thanks Jürgen.
Yes, you may show dissociation.
However, especially if you deal with assembly, then it might be
difficult, if not impossible, to tell your exact subunit composition if
you runBNP only in the first direction. Jacob mentions the possible
occurrence of complex assemblies (AB, BB,
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Dear Jacob,
somewhat adding to list of 'not really answering your question', here is
the reference for native page that still uses a dye thereby trying to
limit the influence of the charge on the speed. Might be helpful as they
discuss some applications. Otherwise AUC really sounds like the
I believe that Francois is correct in saying that a
feature of intel compilers is that they do not work
well on non-intel CPUs. Can't imagine why that would be.
If you have a an intel CPU then using an intel compiler
would be an advantage. Code optimization is usually
architecture specific, so
Hello All-
We have an opening at QuantumBio Inc. for an Industrial Post Doc (or other
early career) in Xray Crystallography Software Development. Below is the short
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You absolutely right, I thought about it.
Maia
Marius Schmidt wrote:
Interestingly, Maxwell's demon pops up here, wh... ,
don't do it.
If you change the reaction rate in one direction 1000 times slower
than
in the other direction, then the reaction becomes practically
That's interesting. Thanks.
Maia
Nadir T. Mrabet wrote:
Maia speaks about native PAGE for which protein mobility (migration)
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a
2D gel: Native in the
Hi All,
I am new to protein crystallography. I would like to know the steps involved
in solving a MAD dataset by using the program in CCP4 where you determine
the phases and then obtain the trace. The dataset is collected at 3
different wavelengths (peak, inflection and remote) using Se-Met as
Hi All,
I am new to protein crystallography. I would like to know the steps involved
in solving a MAD dataset by using the program in CCP4 where you determine
the phases and then obtain the trace. The dataset is collected at 3
different wavelengths (peak, inflection and remote) using Se-Met as
CCP4 way:
locate the Se sites with SHELX (if you use the CCP4I gui it's technically a
ccp4 program :-) )
Try using only your peak data set first. If you can't locate your sites with
the single wavelength then add remote (DAD) and if that doesn't work go MAD.
non-ccp4 way
run hkl2map as frontend
First, you will need CCP4 installed and set up properly. You will also
need to know your protein sequence. Put the latter into a text file
(FASTA format will do).
Next, download the file Elves from:
http://ucxray.berkeley.edu/~jamesh/elves/download.html
then type:
chmod a+x Elves
Elves
Dear CCP4bbs,
I am dealing with a case involving pseudo-translational symmetry.
I wanted to know what was the simplest way to draw NCS copies of a
molecule deduced from the positions I observed in native Patterson. Is
there a translate option where on can give fractional coordinates in
Coot
Compare these two lines from phenix.refine:
REMARK 3 NUMBER OF REFLECTIONS : 46001
REMARK 3 FREE R VALUE TEST SET COUNT : 2339
with those from refmac, ostensibly using the same data and start pdb:
REMARK 3 NUMBER OF REFLECTIONS : 43672
REMARK 3 FREE
Phil,
I think the PDB documentation in this area (such as it is) is unclear,
and it's not easy to fathom what was the original intent. The first
line you refer to:
REMARK 3 NUMBER OF REFLECTIONS :
appears in the section with the sub-heading:
REMARK 3 DATA USED IN
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How about using static light scattering to determine the actual molecular
weight or dynamic light scattering to measure the diameter of the complex.
Sheemei
From: Jürgen Bosch jubo...@jhsph.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, 19 May 2010 11:00:24
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