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SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY
AT THE UNDULATOR BEAMLINE X06SA AND THE SUPERBENDING MAGNET
BEAMLINE X06DA AT SLS FROM MAY - AUGUST 2008
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Bart Hazes wrote:
Dale Tronrud wrote:
[EMAIL PROTECTED] wrote:
> Rotational near-crystallographic ncs is easy to handle this way, but
> what about translational pseudo-symmetry (or should that be
> pseudo-translational symmetry)? In such cases one whole set of
spots is
> systematically wea
Hi Kendall,
As to (1) - it's a hard task. Redox potentials of Cys residues vary with
their environment, so it is very difficult to predict what might happen in
any specific case. Mixtures of reduced and oxidized GSH are commonly used to
maintain a specific redox environment, however if you're real
Hi,
I would like to point out, for the sake of fairness, that thrombin (high
quality bovine thrombin such as sold by HTI for example) is still much
cheaper *to use* than commercial TEV. One milligram of TEV, TVMV, AcTEV, and
so forth can be used to cleave anywhere in between 10 to 100 mg of
're
Dale Tronrud wrote:
[EMAIL PROTECTED] wrote:
> Rotational near-crystallographic ncs is easy to handle this way, but
> what about translational pseudo-symmetry (or should that be
> pseudo-translational symmetry)? In such cases one whole set of spots is
> systematically weaker than the other se
Title: [ccp4bb] protein crystallography position at Nottingham University
Postdoctoral Research Fellow in Protein Crystallography
Applications are invited for a postdoctoral scientist to join a team investigating the structure of coagulation factors and platelet receptors. The project is
Clarification-
Someone wrote:
Ah- that's going way to fast for the beginners, at least one of them!
Can someone explain why the R-free will be very close to the R-work,
preferably in simple concrete terms like Fo, Fc, at sym-related
reflections, and the change in the Fc resulting from a step of
[EMAIL PROTECTED] wrote:
> Rotational near-crystallographic ncs is easy to handle this way, but
> what about translational pseudo-symmetry (or should that be
> pseudo-translational symmetry)? In such cases one whole set of spots is
> systematically weaker than the other set. Then what is the
> "t
Rotational near-crystallographic ncs is easy to handle this way, but
what about translational pseudo-symmetry (or should that be
pseudo-translational symmetry)? In such cases one whole set of spots
is systematically weaker than the other set. Then what is the
"theoretically correct" way to cal
Have you done both experiments at the same temperature? If cryo - have you used
the same cryoprotectant?
same ionic strength? Did mutations cause changes in the surface charge
distribution/hydrophobic/hydrophilic/ pi...pi interactions - if yes how it can
effect quaternary structure of native. Do
In such cases, we always define the test set first in the high-symmetry
space group choice. Then, if it is warranted to lower the
crystallographic
symmetry and replace with NCS symmetry, we expand the test set
to the lower symmetry space group. In other words, the test set itself
will be inva
I've looked at about 10 cases where structures have been refined in lower
symmetry space groups. When you make the NCS operators into crystallographic
operators, you don't change the refinement much, at least in terms of
structural changes. That's the case whether NCS restraints have been appl
Dear Yang,
Thanks for the reply. Both are P21. Point mutant. MR solved the
mutant using native as a model.
What about the crystal contacts of the region that changed then? Is
it different in "native" and "mutant" forms? Both space groups are
the same, but what about the cell dimensions?
Back in the old days, when I worked on crystal structures with 15 or
20 atoms or so, the symptoms of missed crystallographic symmetry
included instability of the refinement, high correlations between
parameters, and (relatively) large deviations between equivalent bond
distances and bond an
Dirk Kostrewa wrote:
Dear Dean and others,
Peter Zwart gave me a similar reply. This is very interesting
discussion, and I would like to have a somewhat closer look to this to
maybe make things a little bit clearer (please, excuse the general
explanations - this might be interesting for begin
Terese Bergfors will be organising her famous Uppsala University Practical
Protein Crystallization Course again, from 15-19 September, 2008. For more
information, see the course website at:
http://xray.bmc.uu.se/terese/course/course.htm
--dvd
*
Yongfu,
Small peptide tags usually don't interfere with protein folding and linker
is not required. With removable tags, the protease site also serves as a
linker. In most cases, putting a protease cleavage site such as TEV or
PreScission site is good enough. The proteases (HRV3C and TurboTEV)
I would export the protein to the oxidative periplasm or alternatively oxidize
the cys-cys-bond with Cu.
Best wishes
Kornelius
On Fri, 8 Feb 2008 10:44:21 -0500
Kendall Nettles <[EMAIL PROTECTED]> wrote:
> I'm trying to engineer a disulfide bond into a protein that has several
> other cysteine
There are strains designed to provide a less-reducing (more
oxidizing) environment (Origami or any of it is "gami" derivatives
from Novagen).
They are deficient in the thioredoxin and glutathione reductases (I
think I recall...I didn't look it back up).
We've used them with good success f
On Fri, 2008-02-08 at 10:44 -0500, Kendall Nettles wrote:
> Also, can I expect 100% disulfide formation from standard bacterial
> expression (assuming good geometry of the cysteines)?
No, E. coli cells are a reducing environment.
-
=
I'm trying to engineer a disulfide bond into a protein that has several
other cysteines.
My question is whether there is a crystallization friendly reducing agent
that can be used to prevent oxidation of the free cysteines without breaking
the disulfide?
Also, can I expect 100% disulfide format
When you say that "NCS is only a local, not global, crystal symmetry"
what you actually mean is that "NCS is only a local not global *group*
symmetry", i.e. NCS symmetry obviously *is* global if symmetry is
defined (as in physics) as *any* operator which superposes (more or
less) 2 copies of the mo
Hi Yongfu,
An interesting approach is to add a c-term biotin tag as a bio-sensor.
If the c-term of the target protein is accessible the biotin tag will
get biotinylated by BirA (co-expression may be necessary depending on
the expression system). You can then easily detect the target protein
Dear Dale,
Am 08.02.2008 um 10:27 schrieb Dale Tronrud:
I'm afraid I have to disagree with summary point (i): that
crystallographic and noncrystallographic symmetry are incomparable.
Crystallographic symmetry is a special case of ncs where the symmetry
happens to synchronize with the lattice
It is hard to say exactly - do you mean the mutant "dimer" is not exact
ie - one chain different to native, and one the same?
Eleanor
yang li wrote:
Dear All,
I have a protein which has the function unit as a dimer. I got two
structures of it. One is the native structure, one is the muta
I'm afraid I have to disagree with summary point (i): that
crystallographic and noncrystallographic symmetry are incomparable.
Crystallographic symmetry is a special case of ncs where the symmetry
happens to synchronize with the lattice symmetry. There are plenty
of cases where this synchroniz
To add to Kay's announcement...
There is also going to be another, "official", ccp4 wiki, distinct from
Kay's wiki, and fulfilling a different purpose. We're in the early
stages of developing it at the moment. The precise roles will probably
evolve over time, but currently we see them as follows:
Dear Dean and others,
Peter Zwart gave me a similar reply. This is very interesting
discussion, and I would like to have a somewhat closer look to this
to maybe make things a little bit clearer (please, excuse the general
explanations - this might be interesting for beginners as well):
1)
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