Ouch!
I completely agree re: single proteins, but I had always found/heard that
protein-protein complexes tolerate freezing/thawing less-well that their
individual components.
Irrespective of generalisations, In the posted case, where apparently,
before freeze complex is intact and concentrated,
Hi All,
I have a simple question about the complex formation between macromolecular
complex and antibody. My protein is stable in the presence of the 5mM DTT
and under these conditions the reducing environment is too strong for the
antibody to survive. I am also now trying to check the
Hello again,
I still think it depends on the protein complex, but I agree that the
consensus/ hearsay/ anecdotal/ old crystallographer's tales revolve around
complexes not working as well as single proteins for freezing. I'm not sure
that has been shown to be the case, although again I would
Jan,
Are you dealing with a whole IgG or an Fab?
Most Fab-antigen complexes will not be affected by 5mM DTT.
Fab production makes use of reducing agents that allow the
antibody hinge region to become exposed to proteases.
If you need DTT to prevent your antigen from aggregating, try
to
From your description, the protein concentration dropped from 10mg/mL
to 1-2mg/mL after freeze-thaw. It's hard to imagine that your protein
has been degraded. Degraded by what (protease)? At -80oC? Did you see
a ladder of lower bands on the gel after the freeze-thaw?
If you're interested
I have to agree that protein is denaturing or precipitating
irreversibly during the freeze-thaw cycle. While HOW you freeze does
matter (cooling slowly in a freezer is almost always destined to fail)
it should be noted that many proteins simply won't tolerate freezing.
(I seem to specialize in
Hi Jan,
you might mutate the Cys residues which are oxidation sensitive,
you could block the Cys thiols in your oxidation sensitive protein,
or you try crystallization at slightly acidic conditions where the Cys
thiol should be more stable (might be bad for the ab-protein complex),
or try
A couple more thoughts:
1. thermodynamics says that proteins denature at low temperatures just as they
do at high temperatures.
2. flash-cooling does away with some of what thermodynamics says (not an
equilibrium process anymore)
3. Whether a given protein can be frozen needs to be
On Wed, 2010-04-21 at 17:21 -0700, James Holton wrote:
The 0.3% chance of a peak being above 3
sigmas assumes that the histogram of electron density values is
Gaussian. It is not! In fact, it is a funny-looking bimodal
distribution (the peaks are protein and solvent regions).
Indeed!
Lots of things change with temperature. An excellent book on the
subject is P. Dohzu's Cryobiochemistry (1977) Academic Press, which I
think every lab that freezes biological molecules should have on hand
($15 on Amazon.com). For example, did you know that the pH of a tris
buffer jumps from
Yes, there has been a conflation of the standard deviation and the
r.m.s. of the distribution when it comes to sigmas. The mathematical
formulas look similar (for a Normal distribution) so some people have
sloppily transferred the meanings of the mathematical symbols from one
concept to the
Hello again.
At first I was not worry but maybe now I am. I have completed a structure and
submitted to the PDB. They queried my Rsym value in the highest resolution bin,
2.5-2.37A (may I dare say it 100%). I was not worried at the time as I had:
99.4% completeness
Mean(I/sdI) of 2.5
and a
Thanks to all who responded. 180 degrees flip of the problematic NAGs, did help.
At the moment, there is no substitute for knowledge when building
carbohydrates - it would be a substantial improvement I think if someone
added intelligent carbohydrate validation tools into Coot.
If you have a
R-statistics are unstable for weak data (such as systematic absences).
Ignore R-sym for your highest-angle bin.
I/sdI is more informative, and from the look of yours I would say you
can reduce your resolution in the sense that you probably have useful
data (I/sdI 1) to better than 2.5 A.
Yeah, stop worrying! Your I/sdI is all that matters.
phx
On 22/04/2010 18:06, Daniel Bonsor wrote:
Hello again.
At first I was not worry but maybe now I am. I have completed a structure and
submitted to the PDB. They queried my Rsym value in the highest resolution bin,
2.5-2.37A (may I
hello everyone
I am about to setup a 3D system for crystallography. right now I can only think
of coot and pymol software. so I might just go with Windows sytem, plus the
Nvidia 3D vision kit
I know there is a detailed list of hardware recommended. but are they
compatiblility issues?
Yeah, but how was I/sdI determined? Most programs allow you to multiply
your sdI by any number you want which in turns means that you can create
any I/sdI that you want.
A multiplicity of 11 does not explain a high Rsym to me.
Jim
On Thu, 22 Apr 2010, Frank von Delft wrote:
Yeah, stop
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If someone has an interest in the following position listed below,
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Takeda
Dear sugar loving people,
I have been working with a lot of glycoproteins (up to 30 %
carbohydrates) at resolutions as bad as 2.8 Å. Nevertheless, I was
able to built sometimes about 10 sugar moieties/carbohydrate chain.
Although, sugar molecules usually have a somewhat bulky density, and
Hi,
I am about to setup a 3D system for crystallography. right now I can only
think of coot and pymol software. so I might just go with Windows sytem,
plus the Nvidia 3D vision kit
If you're only going to use coot and pymol then perhaps a zalman will do?:
There are plenty of structures in the database with R-sym=0.99.
But something is odd here. If I understand R-pim, it should
always be bigger than Rsym, because this factor of sqrt(N/(N-1)) is always 1
Are you saying Rpim is .30 and Rsym is 1.00?
Last time I deposited a structure, Rsym and Rmerge
I am trying to resist answering ccp4bb ... but
Thanks to all who responded. 180 degrees flip of the problematic NAGs,
did help. If you have a poor density (which I guess, generally is the
case for large glycoprotein structures) you have to depend on trial
and error strategy to get the
Hello,
I installed coot-Linux-i686-ubuntu-8.04.3-gtk2-python on my Latop (i686, Debian
testing). Upon start-up, I read the error message:
Error loading icon: Couldn't recognize the image file format for file
Hello Gabriel,
I installed librsvg2-common. Now the error message is gone and the icons are
all complete. Thank you very much!
Tim
On Thu, Apr 22, 2010 at 04:53:27PM -0400, gbirr...@bidmc.harvard.edu wrote:
Hi Tim,
This problem came up on Coot for Mac some time ago. Can you check the
Hi,
While setting up microbatch trays (under oil crystallization),
1. Has anybody used mineral oil (sigma M8410) and obtained some crystal
hits?
2. Has anybody used anything other than Al's oil from Hampton, as Parafin
oil from sigma or silicone oil from sigma and obtained some crystal hits?
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