Hi All,
I want to use a thermofluor for the thermal shift assay. My proteins are
cytoplasmic truncations of membrane protein. I have read about ANS,
sypro-orange and CPM. Which is the once that is popularly used by the
crystallographers for condition optimization for crystallization ??
I have read
Dear Anita,
here is a link to a talk with recommended literature:
http://thermofluor.org/resources/Maksel_Protein-Stability.pdf.
Probably, you will find there the answers you need.
I recommend to try several dyes if available, due to the different
behaviour of proteins and no general rule for
Hi,
The closest example I can think of is the extra density that was found in the
groove of the MHC class I structure (Bjorkman et al., long time ago) which
turned out to be a peptide that was co-purified. As we all know, it turned out
to be one of the major findings of this study as it pointed
Hi all,
Just a (naive?) question: how does one manage to introduce (and deal
with) improper non-crystallographic symmetry in DM ? Or does one has to
go to DMmulti for that (because, by definition, going from one crystal
form to another crystal form is improper NCS) ?
Ta,
F. Vellieux
If you have a CD available (not the one with music on it) you don't need a dye
just sufficient protein and you can thermal denature your protein assuming it
contains some secondary structure elements.
Jürgen
On Jul 19, 2012, at 4:26 AM, anita p wrote:
Hi All,
I want to use a thermofluor for t
It isn't that your space group is wrong, but are you sure that your mtz file
has that space group in its header?
MR will test all possible alternatives - in this case P3121 or P3221 - but
won't change the symmetry information in the input mtz.
You need to do that with a utility like
mtzutils h
My understanding is that the advantage of the thermofluor assay is that
you can test many conditions rapidly unless of course you have some kind
of high throughput CD spectrometer in mind.
> If you have a
CD available (not the one with music on it) you don't need a
> dye
just sufficient protein
Hi Leonard,
I would treat this like a MS/MS ion search experiment looking for the peptide
via MASCOT or similar. Talk to some local Mass Spec people, I'm certain they
could identify it and then you'd be closer to an explanation.
John.
Dr. John Hinks
Syngenta
Jealott's Hill International Res
Dear All,
We are pleased to announce the CCP4 Fukuoka Workshop. All details can be found
at http://www.ccp4.ac.uk/schools/Japan-2012/
Title:
"CCP4 Fukuoka school: From data processing to structure refinement and beyond"
Dates: October 29 to November 2.
Site: Biomedical Research Station, Kyushu
Yes that is very true. But I assume true membrane proteins exclude high
throughput :-)
However the cytoplasmic part might be fine in this case and then I would just
go for Sypro Orange.
Jürgen
On Jul 19, 2012, at 10:39 AM, Edwin Pozharski wrote:
My understanding is that the advantage of the
Anita, In terms of the basics:Thermodynamic stability of a protein is related
to Gibbs Free Energy of unfolding. The Gibbs Free Energy is made of
temperature, enthalpy and entropy (Delta G = Delta H - T Delta S). At Delta G
equivalent to zero, the concentration of folded protein is equivalent
Dear all,
I'm dealing with anisotropic diffraction of a membrane protein.
I've read from previous threads that it is better to not cut off any
data, and that aimless doesn't anisotropically scale the data any way.
So my question is: given my anisotropic diffraction, what resolution
cutoff do i
Our setup is an Olympus SZX-10 with both 1x and 2x objectives and 10x
eyepieces. It's nice having the different objectives for different things--a
close up look at a tiny crystal requires the 2x, but the 1x is less bulky and
has better depth-of-field, so it's better for freezing crystals.
The
Hi
Yes i did check my MTZ file header. It shows p3221. For the same reason i
reprocessed the data again in both p3221, p3121 and p321. Except for p3221
none of the other space groups fit the model. The packing looks good and
the map shows side chains very clearly. When i add the respective side
ch
1) Check free R flag. It may be related with free flag being 1 instead of 0. If
it is the case then you can use newer version of refmac:
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
or from ccp4 v 6.3
2) Try arp/warp
Garib
On 19 Jul 2012, at 18:29, Deepthi wrote:
> Hi
>
> Yes i did check m
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight
for crystallization trials of a soluble protein? Looking for high molecular
wt. protein ~50kDa.
James.
Colleagues,
Does anyone use a local service on an AKTA explorer (or other models)
machine ideally in the east coast or in Maryland? GE Healthcare will stop
supporting this model in a few years, and I am not sure if they are willing
to cover an old machine. I remember someone recommended a local se
I don't think there is such a rule, but in the old days, when we only had
Hampton Screen I and II, the rule was:
- Set up screen 1, look at the drops and you should expect some kind of
precipitation in 50% of the drops. If much less than that, increase your
protein concentration. If much more t
This is almost exactly our basic approach, too. Before we got a
dropsetter, we did 24 wells (1/2 screen) to get a feel for the correct
protein concentration. Some additional rules of thumb we use:
1. We usually start at 10 mg/mL protein and go up or down from there
depending on the results o
Hi James,
You can get the PCT (Pre-Crystallisation Test) from one of the more
famous manufacturers of crystallography products - essentially you can
quickly screen your protein to get an idea if it is too dilute, too
concentrated, or somewhere in the middle. Of course, the results are
representati
Hi Vincent,
my advice is: if in doubt, scale at the highest of those resolution choices.
This way you don't have to go back to scaling later. There is no harm in using
the highest resolution. Don't let yourself be fooled by Rmerge.
best,
Kay
On Thu, 19 Jul 2012 17:52:14 +0200, vincent Chaptal
Hi Narayan,
there's nothing wrong with using data with I/sigmaI 2.5, Rsym 224.3 % for
multiplicity 7.8 and completeness 98.2 %.
However, when you discarded frames you might have made the data worse - one
should only reject data if they deviate systematically (e.g. from radiation
damage). Weak
On Tue, 17 Jul 2012 06:26:39 +, LEGRAND Pierre
wrote:
> Hi Jason,
>
> To answer your initial question about overlaps versus finer slicing, you can
> get a good description of the problem in Fig10
> of Z. Dauter article "Data-collection strategies" (open access article here:
> http://jou
Hi Jason,
I looked at the part of FRAME.cbf you posted, and I'd say it looks ok (i.e.
nothing to worry about).
Some overlap of a reflection doesn't matter much; if its full profile is not
available then INTEGRATE replaces the missing intensity by an estimate based on
the known fraction of the
hello everyone
I just noticed my model would give two very different Rfree
values from refmac depending which mode I use to refine it. Rigid
body it gives a Rfree value of 24.57% while restrained refinement
gives a value of 27.96%. Both are processed at 3.1angstrom
resolution. I am conf
Hi everyone,
I am studying the catalytic mechanism of an enzyme. My structural data
indicates that an Asp is of most possibility to sever as a general base.
The Asp residue is coordinated to a zinc ion. Would it be possible
that the side-chain
carboxyl group of Asp attacks a carbon atom of the sub
Norman,
the value of free-R alone doesn't tell a whole lot, though the numbers you
quote seem good given the resolution. What's Rwork?
The behavior of free-R you tell is also totally expected: in rigid-body
refinement there are way less refinable parameters!
Most likely you should proceed with ind
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