Dear All,
I am working on protein refolding via dialysis in large volumn
(typically 2~4 litters). It was problematic when I wanted to concentrate the
solution to at least less than 500ml. If you know any device appropriate for
such task, please help me out:)
Thanks in advance!
Sincerely,
Xuan
Dear Chris,
The answer depends on why you want to exclude oxygen for crystallisation
and how many screens you want to do. Did you purify your protein in the
absence of oxygen?
If you only want to set up a few screens for a single construct - I
would suggest you put the screens and the plates in
Hi, Xuan Yang
I know a device from PALL Corporation, which is used to concentrate large
volume of solution by passing through a filtering film. A similar product
from GE is used in our lab, too. You can contact these two companies for
details.
Sincerely yours,
mrmar
E-mail:
Yuan Cheng wrote:
Eleanor Dodson wrote:
This phenonema doesnt necessarily mean you have lattice-tranlation
defects - pseudo translations are quite common with perfectly good
crystals.
Lattice translation defects usually imply your crystal has two or
more layered different crystals in the
Dear all,
Recently I am secretely expressing an ectodomain of a human membrane protein in
insect cells. The expression level is moderately high but the target band in
the SDS-PAGE gel seems smearing and this is cosistent with the western blot
results. Does it result from the heterogeneous
Can you put a 6His-tag on your protein and add some Ni-NTA beads to
your solution to capture (and concentrate) the properly folded
molecules?
Quoting Xuan Yang pattisy...@gmail.com:
Dear All,
I am working on protein refolding via dialysis in large volumn
(typically 2~4 litters). It was
This means that some U tensors are non-positive definite (i.e. no nice
thermal ellipsoid). If by transient, you mean they have gone away by the
last cycle of refinement, then you can maybe ignore them. But probably,
you need to adjust your restraints, e.g. the SPHEricity restraint (under
Geometric
Dear Dr Ku,
It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg
and 1mM EDTA.
And I want to try other affinity columns, just don't know what resin would
be appropriate.
Sincerely,
Xuan Yang
2009/9/3 Shao-Yang Ku s...@embl-hamburg.de
Can you put a 6His-tag on your
Dear Xuan Yang,
for such large volumes we use a Pellicon type system. The one we have in
the lab is a polyethersulfone PTJK prep/Scale TFF 2.5 FT2 High flux 10 k
from Millipore.
good luck
Demetres
Xuan Yang wrote:
Dear All,
I am working on protein refolding via dialysis in large volumn
Dear all,
does anyone have experience on how to convert a raw-data file from SAINT into a
mtz-file, suitable for input into SCALA? I tried combat (saint option), but the
reflections are apparently already reduced to the asu and I'm not sure if the
program picked up all the necessary parameters
Dear all,
I just solved my crystal structure and I was looking for my ligand. I have 4
molecules in the AU, but I found just in one molecule density for my ligand. In
the other 3 molecules appeared deep red density when I tried to build
something. Is this generally possible? The binding sites
dear marek
although your description is not usual, we have seen this in some cases and
with different systems here. this behaviour is also not unheard of in the
literature (for example: menetrey et al., JBC 277, 30950 (2002). I would
continue pragmatic and build the ligand where the difference
Deal all,
Sorry for the non-ccp4 query once again.
I need suggestions regarding the improvement in crystal quality. I have
crystallized a protein in MPD. The crystals grow like a thin plates and the
plates are stacked together. So, the mosaicity is very high and also
indexing is difficult even at
James,
I think the standard suggestions apply. Try to tweak your
crystallization conditions to get a single crystal (i.e. not a series of
stacked plates). PEG 400 can be a good substitute for MPD, but you
could also try additive screens as well as co-crystallization with
products,
We had a similar situation: great diffraction,
multiple lattices due to stacked plates that appeared to be single
crystals. We ran an additive screen with about a dozen common salts and
solvents, e.g., AmSO4, LiCl, MgSO4, AmPO4, PEG400, EG, glycerol, DMSO,
etc. at 100 mM for salts or 5-10% for
Marek,
Your observation is not unusual. We have seen it for cases with 2
independent molecules in the ASU (Mulichak et al. Proc. Natl. Acad.
Sci. USA 100, 9238-43, 2003.) and a symmetric 222 tetramer in the
ASU (Mulichak et al., Biochemistry 41, 15578-89, 2002). Continue
refinement,
An anion exchange column with a large enough bed volume might be used to
concentrate the protein. You probably need to change the pH of the
solution so that the protein sticks to the column.
On Thu, 2009-09-03 at 17:05 +0800, Xuan Yang wrote:
Dear Dr Ku,
It was a wonderful idea! However,
Hey there Chris,
Additionally, I have seen trays set up under Argon atmosphere in glove
bags. They are sealed and left in the bag until inspection where they are
temporarily brought out and returned.
Then, when harvesting, they would use a glass dish (taller than the tray
plus some working
1 week is left to the deadline for applications to
the Practical Course on Training in methods for Macromolecular
Crystallography M2M-2009: From Measurement to Model.
The course will take place at the EMBL Hamburg Outstation, the DESY
synchrotron site during the period:
Wednesday October
Hi Eleanor,
Are you sure about the application of twinning tests to look for
lattice-translocation defects? In twins the intensities sum, but in
lattice translocation cases, the domains can be so small that you can't
simply sum intensities . . . at least that's how I interpret
1. Yes, Ion Exchange (not just Anion as we don't know anything about
their protein (unless I missed an email)) is a great capture step if you
have the time to mess about.
2. When I have been refolding recently, after the refolding step we have
found that a good, old-fashioned Ammonium sulphate
Have you tried using these crystals to produce seeds for matrix seeding?
charlie
Deal all,
Sorry for the non-ccp4 query once again.
I need suggestions regarding the improvement in crystal quality. I have
crystallized a protein in MPD. The crystals grow like a thin plates and
the
plates
On Thu, Sep 3, 2009 at 1:33 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote:
Lattice translation is effectively one form of twinning, you can visualise
it as a set of crystals where that lattice are aligned in 2 dimensions but
there is slippage along the third. So each reflection is in fact the
Dear all,
I have a Se-Met crystal diffracted to 5.6A and a full data set of the same
native crystal diffracted to 2.8A. Since I can hardly improve the quality of
the Se-Met crystal, may I ask some suggestion from the community about am I
able to solve the structure based on the above
Dear Zhen,
you could try Santosh Panjikar's Autorickshaw server at
http://www.embl-hamburg.de/Auto-Rickshaw/
to see if it finds a solution.
Even risking to suggest something you already did or cannot carry out for
some reason:
Have you tried micro-seeding of the Se-Met drops with the
My suggestion would be to attempt finding Se atoms in the MAD dataset and
then use these coordinates to do sulphur phasing - assuming of course that
you can get the native crystals to diffract at an appropriately low-energy
X-ray source.
Artem
Dear all,
I have a Se-Met crystal diffracted to
Dear Linux/laptop dudes,
I plan on turning a stack of older XP laptops into linux computers for
crystallography workshops. Any recommendations based on experience which
package to get with best support for laptops? Also, ease of dual boot
options might be
interesting. Perhaps off-board.
Hi,
Yes �C it’s quite likely that your protein is glycosylated. No worries,
it’s not a death knell �C in fact glycosylation can be interpreted as a
good sign :-)
Crystallization: yes, this may be an issue. On the other hand it could also
be not a problem. Try crystallizing the protein
If one of its eigenvalues of the ANISOU matrix is negative, the matrix is
non-positive definite
leading to the MAKE_U_POSITIVE problem.
I understand that this could indicate that an atom is sitting on a saddle point
between two minima and that the atom could be split into two conformers. I had
Based on my experience, ubuntu has the most hardware drivers. If your
computers are old, they should be supported by most linux distribution
versions. CentOS may also be a good choice. Dual boot would not be a
problem.
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
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