We've been using the S219V mutant for cleaving out tags. We usually do our
cleavage reactions in an overnight dialysis after a Ni-column into 50mM Tris,
100-200 mM NaCl, 5 mM BME at 4C. I've never found problems with losing any
protein in the reaction and recover usually 90% of the protein
Agitation can cause denaturation of proteins resulting in loss of
activity, precipitation and even cross-beta amyloid fibre growth.
Partial unfolding will probably make most proteins more protease sensitive.
Alun.
On 31/03/2011 20:41, gauri misra wrote:
Just an offshoot of the same
Hi Ethan,
Awsome progress! Really, I looked for other options like such. 2011
will be a good year for crystallography. I should implement this in PDB_REDO.
Cheers,
Robbie
Date: Thu, 31 Mar 2011 23:06:47 -0700
From: merr...@u.washington.edu
Subject: [ccp4bb] Crystallographic Breakthrough
Dear Ethan,
I would really really like to enhance all my PDB files, but I am
concerned I will create a black hole in my hard drive. I hope you can
convince me of the safety of your tool, thx
Flip
On 4/1/2011 8:06, Ethan Merritt wrote:
Hi to all on ccp4bb:
What better day to announce the
Maybe the next version of coot as well as pymol/ Raster3D could also display
virtual particles. This would really flashily push the quality of our models,
especially on the title pages of the electronic versions of journals. There
could even be a special July-14th-mode
I'm pretty sure Coot has been displaying it all along. In the early
days it displayed it much better, I must say, which is why it tended to
crash.
Maybe the next version of coot as well as pymol/ Raster3D could also display
virtual particles. This would really flashily push the quality of
Hi
I'd just process it in iMosflm, and run the Quickscale task after integration.
With almost no effort you should get a rapid visual indicator (in the graphs
produced by Scala) of the discontinuities between the wedges.
If the discontinuities are too big, then you might encounter some items
Have you noticed the date ? It's April 1!
At 10:31 01-04-2011, Frank von Delft wrote:
I'm pretty sure Coot has been displaying it all along. In the early
days it displayed it much better, I must say, which is why it tended to crash.
Maybe the next version of coot as well as pymol/ Raster3D
Well played, Ethan!
At 07:06 01-04-2011, Ethan Merritt wrote:
Hi to all on ccp4bb:
What better day to announce the availability of a breakthrough technique
in macromolecular crystallography?
Given recent discussion and in particular James Holton's suggestion that
the problem of disordered
On Fri, Apr 1, 2011 at 5:30 AM, Santarsiero, Bernard D. b...@uic.edu wrote:
Ian,
I think it's amazing that we can program computers to resolve a b c
but it would be a major undertaking to store the matrix transformations
for 22121 to 21212 and reindex a cell to a standard setting.
I think
The program appears a bit black-box to me, could you provide more details
(today of course).
Mark
Sent from my HTC
- Reply message -
From: Robbie Joosten robbie_joos...@hotmail.com
Date: Fri, Apr 1, 2011 11:04
Subject: [ccp4bb] Crystallographic Breakthrough - DarkMatter Version 1.0
Excuse my naive (perhaps ignorant) question: when was the
abc rule/convention/standard/whatever introduced? None of the
textbooks I came across mentions it as far as I could see (not that this is
reason for or against this rule of course).
Thanks,
Boaz
Boaz Shaanan, Ph.D.
Hi Frank,
I described in the previous e-mail the probabilistic interpretation of
B-factors. In the case of very high uncertainty = poorly ordered side
chains, I prefer to deposit the conformer representing maximum a
posteriori, even if it does not represent all possible conformations.
Dear Gerard,
I agree with you based on debates at some conferences.
But, based on what I have seen here so far, it seems to me that everybody knows
exactly what to do with disordered side chains.
People that want to build structures to best fit the data tend to prefer
omitting disordered side
Hi Robbie
If it's probability you're after, if there's no density to guide you
(very common!) you'd have to place all likely rotamers that don't
clash with anything, and set their occupancies to their probability (as
encoded in the rotamer library).
Which library? The one for all side chains
I assume TLS is supported or do we have to wait for version 1.1 ? When will you
have a 10.7 (lion) standalone version compiled ?
Jürgen
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry Molecular Biology
Johns Hopkins Malaria
On 03/31/11 18:08, Patrick Loll wrote:
We've just collected a number of inverse beam data sets. It turns out the
crystals showed little radiation damage, so we have a lot of data: 2 x 360 deg
for each crystal, broken up into 30 deg wedges. The collection order went like
this: 0-30 deg,
Hi Ethan,
many thanks for that - your Dark Matter really (en)lightened my day! I
wonder, how many pdb records in the future will contain the
corresponding remark lines that your incredible perl script produces :-)
Best regards,
Dirk.
Am 01.04.11 08:06, schrieb Ethan Merritt:
Hi to all on
Dear Boaz,
I think you are the one who is finally asking the essential question.
The classification we all know about, which goes back to the 19th
century, is not into 230 space groups, but 230 space-group *types*, i.e.
classes where every form of equivalencing (esp. by choice of
Do you have a list of dark-matter-aware PDB refinement programs?
Adding dark matter and refining in TNT or Xplor gives me exactly
the same R as without. Furthermore the final refined files have
lost the dark matter as far as I can see. This leads me to believe
these programs are completely
Oh, that is where those pesky inhibitors I couldn't find were
hiding...
On Thu, 2011-03-31 at 23:06 -0700, Ethan Merritt wrote:
Given recent discussion and in particular James Holton's suggestion
that
the problem of disordered sidechains is a problem akin to the
difficulty
of describing
Dear all,
I have been trying to compare a model that I'm refining against the
native SFs using SFCHECK. SFCHECK finishes normally (no errors in log file,
seemingly complete list of output .ps files), but produces a postscript file
with only the first page of output (and it is mostly
Dear Gerard,
The theory's fine as long as the space group can be unambiguously
determined from the diffraction pattern. However practice is
frequently just like the ugly fact that destroys the beautiful theory,
which means that a decision on the choice of unit cell may have to be
made on the
Hi all,
could you use its position in real space as a target for (to make it easy,
rigid body) refinement? Real space meaning the electron density around the
scatterer in an anomalous LLG map.
Some other restraints: Say it's a metal cofactor and you know that it needs to
be in a specific
Will this affect my reprocessing of the data with D*TREK on my journey
to XPLORE the planets MERCURY and rPLUTO in my ENDEAVOUR to find and
BUSTER some CRYSTALS with my on-board TNT into XPOWDER?
I am still trying to GRASP the idea of AUTODOCKing on precise HKL
locations based on the SHARP
Dear Ian,
Well, it *IS* broke. If you are running some type of process, as you
implied in referring to LIMS, then there is a step in which you move from
the crystal system and point group to the actual space group. So, at that
point you identify P22121. The next clear step, automatically by
People of the disordered sidechains, ave!
-
perhaps the IUCr and/or PDB (Gerard K?) should issue some guidelines along
these lines? And oblige us all to follow them?
(Mark J van Raaij)
-
this discussion has flared up many times in the past, and maybe it's time for
a powerful
Well, actually you probably haven't subtracted the dark R value from
your final R. This and other values are available in a dark folder
on your hard drive, which is impossible to see/read, but which
contains a lot of important information which will make your structure
have a much higher impact
Strangely enough, Nature may be the only journal that fully enables reviewers
of protein crystallography to do the proper job - reviewer access is provided
to unreleased coordinates AND diffraction data upon request! (no this is not
April1 joke). There are reasons to suspect that the request is
Hi all,
As part of its recent winter update, the Protein Data Bank in Europe (PDBe;
http://pdbe.org/) has improved its facility that allows for tandem searches of
PDB and EMDB. It was designed to allow users to carry out many of their
day-to-day searches (without the need to fill out a
I need to calculate the electron density values for a list of spatial
locations (e.g. atom positions in a model) using an mtz-file that
already contains map coefficients. To write my own code may be easier
than I think (if one can manipulate mtz columns, isn't the only problem
left how to
If you have break in the protein chain, this problem will occur. Put TER card
in the PDB file where ever the chain break is there, then run SFCHECK. The .ps
output now will be complete and should be able to see in the viewer.
HTH
-Karthik
Dear all,
I have been trying to compare a
April 1st, 2011
Dear Andy,
We have observed the same problem before,
and just today we could finally find an explanation.
Apparently, a new (still undocumented) functionality
was quietly introduced into few widely used oscillation data
processing programs, enabling the recording of the
Ed Pozharski wrote:
I need to calculate the electron density values for a list of spatial
locations (e.g. atom positions in a model) using an mtz-file that
already contains map coefficients. To write my own code may be easier
than I think (if one can manipulate mtz columns, isn't the only
Le 01/04/11 12:39, REX PALMER a écrit :
Dear Protein Crystallographers
I would like to share with you something I came across today.
Unfortunately I was only able to copy the first 4 pages of the article
by MV King as I need to post the email before 12am and the quality of
the copy is
Hi Ed,
if you are familiar with CCTBX then
map_value = map_data.eight_point_interpolation(site_fractional)
Also, there is a similar method that will just give you the density value at
closes grid point.
Let me know if interested, and I can send you a 10 lines Python
script-example that will do
The meaning of B-factor is the (scaled) sum of all positional
uncertainties, and not just its one contributor, the Atomic Displacement
Parameter that describes the relative displacement of an atom in the
crystal lattice by a Gaussian function.
That meaning (the sum of all contributions) comes from
In my experience, the Refmac's default restraints on B-factors in side chains
are too tight and I adjust them.
Concur. See BMC p 640.
BR
This was not so much an advance but a remarkable observation. We have since
learned that these clathrates are entirely impractical. The problem is not so
much their dextrorotatory properties, which are more or less a nuisance, but
that they are too dense and have absolutely no affinity for
I'm not sure I entirely agree with ZO's assessment that a B factor is
a measure of uncertainty. Pedantically, all it really is is an
instruction to the refinement program to build some electron density
with a certain width and height at a certain location. The result is
then compared to the
Congratulations on your amazing discovery, which immediately suggests many new
lines of inquiry:
Does dark matter affect macromolecular stability? Can it explain the
difficulty some students have in sample preparation? Is it found in higher
concentrations in brains that are thought to be
In this case, I'm more on ZO's side. Let's say that the refinement program
can't get an atom to the right position (for instance, to pick a reasonably
realistic example, because you've put a leucine side chain in backwards).
In that case, the B-factor for the atom nearest to where there should
Dear All,
I was polled for a recommendation for a good PCR instrument,
but I am not much of a molecular biology person - if someone could
please help and kindly send some recommendations to
Eric W. Reinheimer ewreinhei...@csupomona.edu
Best regards, BR
Dear Karthik and Sergei,
Thank you for the replies (helpful and humorous). Karthik, I confirmed
that the chain breaks in my .PDB have TER cards, but I arrive at the same
result. Perhaps dark matter is to blame for this singularity after all...
In that case, maybe I just need to try
Dear all,
sorry for being off-topic!
I have been experimenting with the latest phenix build [1].
I am fairly impressed with phenix.canephor [2]. At first sight the projected
density looked a little week until I realised that I had used
phenix.project.mediterranean. As I switched to
On Friday, April 01, 2011 01:51:31 pm Tim Gruene wrote:
Subsequently I attempted to improve the result using phenix.calzone [3].
This program comes in both Chicago and New York localizations.
Do you know which one you have?
Unfortunately this program crashed because of a missing
How *did* those physicists get such a convenient hypothesis, when the
rest of us have only light matter to work with! ...Or do we also
really have our dark matter too?
JPK
On Fri, Apr 1, 2011 at 2:12 PM, Phoebe Rice pr...@uchicago.edu wrote:
Congratulations on your amazing discovery, which
It may simply be the case that all those seleniums scattering anomalously are
pumping the dark matter.
Sent via BlackBerry by ATT
-Original Message-
From: Jacob Keller j-kell...@fsm.northwestern.edu
Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK
Date: Fri, 1 Apr 2011 17:25:02
To:
Personally I think it is a _good_ thing that those missing atoms are a pain,
because it helps ensure you are aware of the problem. As somebody who is in
the business of supplying non-structural people with models, and seeing how
those models are sometimes (mis)interpreted, I think it's better
Hi Ed,
I wrote a short program name HYDENS that takes a PDB file and an H K L
amplitude phase file for a full hemisphere of data. You can make the
latter from an MTZ with sftools. The program is on my website at
http://129.128.24.248/highlights.html. There is a linux executable as
well as
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