Hi All,
I am working on a protein which has a membrane spanning region and as
cytosolic domain.I have made various deletion constructs of the protein, so
that I can have a crystallizable fragment. There is no homologues mentioned
in the pdb for this protein.
All of these constructs are purified
If you do the calculations, you will find that you need a FREE ligand
concentration of >10 * Kd to get >90% occupancy of the binding site.
If you have e.g. a ligand with a Kd of 100 nM, you would need a free ligand
concentration of 1 µM. However, a solution of 10 mg/ml of a protein of 30 kDa,
ha
Once waters have been located and refined is there a program that analyses
their positions
in terms of solvation shells?
Can the results be compared easily with those from related known
protein structures?
Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.home
The Berger laboratory at the European Molecular Biology Laboratory (EMBL)
Grenoble, France seeks to recruit an outstanding postdoctoral scientist in
structural biology with a research focus directed towards the structure of
macromolecular assemblies. The major theme within the group is the stru
We have structures of two states of a large multi-domain protein in
which domain
movements of up to 30 degrees are observed. Is there a programme
available which
attempts to determine rigid-body units and hinge regions in an
'unbiased' way?
thanks for your suggestions,
Stephen Cusack
--
*
Dear Stephen,
I find DynDom very good (http://fizz.cmp.uea.ac.uk/dyndom/)
otherwise the morph server
(http://molmovdb.mbb.yale.edu/molmovdb/morph/) also performs similar
analysis, cheers, Matt.
On 26/08/2011 11:25, Stephen Cusack wrote:
We have structures of two states of a large multi-d
DynDom version 1.5 is part of the CCP4 suite.
There is an interface for it under Program List.
Although Steve Hayward appears to have a new version DynDom3D which we
don't have. No idea if it would be better for your case.
HTH
Martyn
On Fri, 2011-08-26 at 11:28 +0200, Matthew BOWLER wrote:
> De
Frank,
Point #1 - fair point; the reason Rfree is popular, though, is because it
> is a *relative* metric, i.e. by now we have a sense of what "good" is. So
> I predict an uphill fight for LLfree.
>
Why? I don't see any difference. As you say Rfree is a relative metric so
your sense of what 'g
Dear All
I was working on a Human protein and expression and
solubility is good in E.coli and purification is One step (His-Tag), and i
need to cleave the Histag before screens, if not
the protein will precipitated and Aggregated, but after trying for 1.2 years
i have crystals
Depending on how big your broccoli is I would simply crush them and mount as
big of a piece as possible and see how well it diffracts.
I assume you have tried already:
- glycerol
- changing protein:reservoir ratios
- temperature
- adding oil to either / and the reservoir, your drop
-crushed up som
I would definitely try gelfiltration (how do you get rid of the cleaved tag
anyway, sample buffer exchange?) but especially ion exchange. A homogeneous
sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in
ion exchange. Beyond that i would make some point mutations on surf
Hi,
I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and
determined its structure. It turns out that I see several metal sites in the
structure, mostly cadmiums. Is there any information published (preferably a
review) which summarises data on cadmium sites in proteins s
Hi Sandeep, if someone sends one, kindly share the references.
In general, Ca2+ could have more Asp, Asn kind of coordination and distorted
pentagonal bipyramidal geometry with waters (about 2.5A), Cd can also have
S- since it is softer, I guess Co might have N/O/S (i.e all three with
paired elect
Hello Sandeep,
I have been in this situation many times before but with different metal
ions..
I have found papers published by Marjorie M Harding very useful in such
situations. In fact, there are lots of information on-line on which is
available here (including all the references for his papers)
While I believe there is plenty written about metal coordination, the best
approach, IMHO, is to search PDB for metal of your choice at resolution as high
as you can get and compare to your case.
Vaheh
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.
Dear all,
Do you know how to generate some closed density map (mesh) that can wrap
ligand without tangling with electron density map of other residues?
Many Thanks!
Hui
Hi,
I recall seeing some (old) ConA structures with Cadmium substituting for one of
the native metals (Ca+2 or Mn+2). They must be in the PDB database.
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
E-mail: bshaa...@bgu.ac.il
I would calculate an omit map. Delete your ligand and use the resulting
pdb file to calculate a map. Density for your ligand will show up as
positive difference density.
Herman
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of RONG hu
Dear Dr Ian
from your argument i could not understand how many cycles to refine before
submitting the coordinates to the PDB. what is the upper limit 100 or
thousand or million according to my understanding, its more logical to
stop the refinement when over refinement is taking place (when R
I think the number one criterion for choosing a map for a figure should
be "What am I trying to show in this figure?" If you simply want an electron
density mesh that covers your ligand calculate an Fcalc map. This type of
figure is usually worthless in a research paper but may have value in
Hi all,
We have several primitive monoclinic datasets for the same protein with various
ligands, with essentially the same unit cell parameters. We would like to have
these with the molecules/density oriented the same way for easy comparison, but
as chance would have it, some have effectively
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Dear Greg,
with XDS I normally set "REFERENCE_DATA_SET" to the first one indexed /
integrated in order to maintain consistent indexing between data sets.
Not sure whether similar options are available in other integration
programs, but if I remember c
Dear Protein Chemistry (?),
When R and R-free drift off you are probably refining with suboptimal weights.
If anything, it proves you still have work to do. At convergence R and R-free
do not really change anymore so neither does the difference. If you have
already done a lot of rebuilding and
Cadmium(II) prefers softer Lewis bases (e.g.
thiolates and imidazole ligands more than carboxylates) and
is commonly found in a four-coordinate environment, similar to
zinc(II).
Cheers,
--
Roger S. Rowlett
Gordon & Dorothy Kline P
Postdoctoral position at the University of Chicago
The Keenan lab in the Department of Biochemistry & Molecular Biology at the
University of Chicago seeks to recruit an outstanding postdoctoral scientist
with a strong interest in membrane protein biogenesis. The main goal of the lab
is to under
Hi
Yes, in the CCP4 world, pointless is the program for you.
On 26 Aug 2011, at 17:00, Tim Gruene wrote:
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Dear Greg,
with XDS I normally set "REFERENCE_DATA_SET" to the first one
indexed /
integrated in order to maintain consistent indexing bet
Hi AR
Please define what you mean by 'over-refinement' as it's not a term I use:
does it mean 'convergence', or 'over-fitting', or 'over-optimisation'
(whatever that means) or something else?
If by "LLG is stabilized" you mean it has converged then I agree that's a
possible stopping criterion, bu
Hi,
I do have 10% glycerol in my buffers, and still the constructs come in the
void volume.
and I have sarkosyl in the lysis buffer. but none in the elution or dialysis
buffer. So do I still need detergents please suggest.
reg.
Anita
On Sat, Aug 27, 2011 at 12:13 AM, Pius Padayatti wrote:
>
Hi Yury,
I have done dynamic light scattering and it shows its polydispersed.
Please let me know if it is still ok for setting trays.
reg.
anita
On Fri, Aug 26, 2011 at 10:21 PM, Yuriy Patskovsky <
yuriy.patskov...@einstein.yu.edu> wrote:
> Anita,
> an assembly may be quite large - I would check
Hi everyone,
Due to recent downsizing, a complete Rigaku MicroMax-007 system needs
a new home! R-Axis IV++ detector, computer, software and all other
components included. Please contact me off-list for details.
Happy Friday!
Erin
650-804-7008
ress...@gmail.com
While it's not a review on protein binding sites, the following review might be
helpful in understanding its chemistry:
Holloway & Melnik (1995) Main Group Met. Chem. 451-585.
Also, David Giedroc has written a couple of useful reviews that include
discussions of cadmium binding proteins.
Cadmi
Gregory Bowman wrote:
Hi all,
We have several primitive monoclinic datasets for the same protein with various ligands,
with essentially the same unit cell parameters. We would like to have these with the
molecules/density oriented the same way for easy comparison, but as chance would have it,
Dear Crystallographers,
I have ~30 data sets from ligand soaks of my protein of known
structure (all approximately the same cell. Can anyone suggest a high
throughput method which would do molecular replacement, refine, then
output new blobs, perhaps as a water pdb file? I am sure they do this
or
Hi all,
What are the most successful methods you know of for dehydrating a crystal
prior to freezing it? I am trying to push the resolution of my crystals.
Thanks,
Andrea
Hi Greg,
you could also CAD them into one huge mtzfile with different labels e.g.
FP_Lig1, FP_Lig2 etc. Then they would be the way you need them for direct
comparison in Coot, Pymol or whatever program you wish to use.
Jürgen
On Aug 26, 2011, at 11:55 AM, Gregory Bowman wrote:
Hi all,
We ha
See message to Greg :-)
CAD them first, run MR with one and then just refine using different labels,
inspect your difference density maps and enjoy your success rate of 6% :-)
You can certainly write a tiny script which would use different labels and do a
quick phenix.refine or Rafmac.
I would sk
Andrea L Edwards wrote:
Hi all,
What are the most successful methods you know of for dehydrating a crystal
prior to freezing it? I am trying to push the resolution of my crystals.
Thanks,
Andrea
First of all, be aware that not all crystals are improved by
dehydration. Some need to be drier,
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