In addition to reducing the beam divergence, you may wish to use a smaller beam
size by using a smaller collimator or making the slits smaller. A smaller
crystal can also help to spatially separate the Bragg spots as can moving the
detector closer to the crystal. Yes, closer to the crystal.
More accurately: adjustable pin. Their *loops* are all adjustable
(as opposed to Mitegen or MDL).
Those pins are not very useful if your crystal is already mounted. And
once you've bent that pin, it can't be stored again, because robots and
tongs won't close around them.
They do have the
Hi,
Besides aligning the long axis with the rotation axis, which is the most
important, there are a few more things that may help:
1) Try optimizing the freezing to reduce the mosaicity (if not ideal), or shoot
at RT if possible. With higher mosaicity, the shape of the reflections are
my experiences:
C2 with a long axis parallel to the shortest crystal dimension, crystals were
plates. Used prebent loops to fish the crystals. Personally I haven't tried to
bend loops in mounted crystals as Frank does, but it sounds very useful.
bar-shaped P321 crystals with hexagonal
Hi all,
I am having a structure where there is a disordered water .I have fixed one
water into the density, yet there is a positive density about 1.6 ang away.
I want to put in another water and attribute 0.50 occupancy to each of the
the waters while attributing them to one water in reality. I
I would use in coot: calculate - model/fit/refine - add alt conf. This
should generate a correctly defined alternative water.
Good luck!
Herman
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of arka chakraborty
Sent:
In addition to all the excellent suggestions if you can you can also move your
detector away from the center of the beam aka increase the detector size in one
dimension. Not sure if you can do that at your home source though. By moving
the center of the beam say to the lower 9/10 of the
Dear All;
We purified a His tag protein by Ni-NTA and gel-filtration from E.coli.
We tried two endotoxin removal resins from Pierce. However, it is hard
to remove the endotoxins in the purified protein because the protein bound to
the resin as well.
This
Jim Pflugrath wrote:
In addition to reducing the beam divergence, you may wish to use a
smaller beam size by using a smaller collimator or making the slits
smaller. A smaller crystal can also help to spatially separate the Bragg
spots as can moving the detector closer to the crystal. Yes, closer
Hello CCP4bb,
Firstly, thanks to all for your comments. However, I'm still unsure how to
sort all of this riding hydrogen business out.
Robbie's comments seem particularly apt:
Because there were some reporting errors in the past
(http://proteincrystallography.org/ccp4bb/message18808.html)
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Dear ARKO,
you can edit the PDB file by doubling the line of the respective water,
assign the the Alternate location indicator (column 17) to 'A' and 'B'
(see e.g. http://www.wwpdb.org/documentation/format33/sect9.html#ATOM),
and change the
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Dear Paul,
I doubt there is a foolproof way - if the authors did not describe the
refinement procedure properly in their publication and the PDB file
lacks this information in its header, I guess you cannot tell.
Tim
On 03/07/2012 04:00 PM, Paul
Hi Jerry,
We have used 0.05% Triton X-100 in our wash buffer to wash the protein
on column (Ni-IMAC for His-tagged proteins and MabSelect for
antibodies). 20 column-volumes wash followed by 20 column-volume wash
without detergent is typically enough to reduce the endotoxin
significantly.
I would
Dear All:
I try to add water to my model.
Here is how I did:
Coot: Find Wates
Map: FWT PHWT; 1.8 rmsd; Distances to protein atoms: 2.4
min/3.2 max
Coot found 270 water molecules.
I then examed these waters. Most of them had ball shape. Some had two or
more balls together.
Uma,
Remember that your structure, ultimately, is a model. A model is
your best judgment of the true representation of the protein
structure in your crystal. Your model should make chemical sense.
Coot is pretty good at placing waters, but it cannot
I tried to use 250mM Tris pH 7.6, 1M NaCl and 5~10% 2-propanol for
dialysis. It works,
On Wed, Mar 7, 2012 at 6:50 AM, Jerry McCully
for-crystallizai...@hotmail.com wrote:
Dear All;
We purified a His tag protein by Ni-NTA and gel-filtration from
E.coli.
We tried two
*Dear CCP4
*
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*best Preben
*
*
Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership,
University of Oslo*
**
*1 PhD research fellowship*
*Background:*
The Centre for Molecular Medicine Norway (NCMM, www.ncmm.uio.no)
Hi all,
Sorry for the off topic question. Could someone suggest a positive control
for FP assay? I am developing this assay and would like to use it for trouble
shooting an instrument.
Thanks,
Huiming Li
I'd appreciate suggestions about how to reorganize my My DirectoriesProjectDir
listing.
Its currently got several years worth of work and is getting hard to work with.
I'd like to archive old projects (say by year) but keep all the CCP4i files for
each project intact, so that I can easily go
Hi Uma,
Water has the capability of making 4 h-bonds, 2 from the two non-bonding pairs
of electrons (h-bond acceptors - expect an N-H from an amide for example) as
well as the two hydrogens (h-bond donors). I would refine all those waters and
assume they are waters. If the distance to the
Hi, Joel:
Thank you for your comments.
Some of these waters have 4 contacts all with O from adjacent residues.
As O can be doner as well as acceptor, I would consider them as 'real'
water. Some of these 'water' have more than 4 contacts, I
would consider them as 'false'.
I lower the H-bond
Hi
1) refmac's default option has been generate all starting from version 5 (it
is around 14 years). The reason is for most tests generating hydrogens gave
good R or geometry. And if you look at the original paper by Ramachandran (I
think it was pulished in 1963) you see using hard ball
Ø Some of these 'water' have more than 4 contacts, I would consider them as
'false'.
How about bifurcated hydrogen bonds?
BR
I guess we would all like to be able to do that!
High resolution structures show that a) there are well defined H2Os with
tidy H bonds. b) there are multiple networks where the waters (and many
side chains) have partial occupancy c) there is a soup of other stuff
which was in the
Dear Uma,
The water pictured in W12-1.jpg: could this be a potential metal ion? If
you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4
coordination with oxygen atoms. So, provided your crystallization condition
or buffer contains metal ion(s), you could attempt to see if it fits
If you have fluorescein (10-100nM) in your lab, you could measure the effect of
varying the amount of glycerol (0 to 100%) on the fluorescence anisotropy of
fluorescein in a phosphate buffer. The anisotropy should increase with
increasing concentration of glycerol. Fluorescein by itself in a
Welljust to add, it has been our contention that many of the metal ions
have been modelled as waters in several structures- due perhaps to the lack
of sufficiently high resolution data. We published some of the potential
metal binding sites in many structures a few years ago:
Proteins. 2008
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