I reckon you could share hypothetical review comments for educational purposes.
-Original Message-
From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.)
Sent: Thu 10/28/2010 12:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst
It is instructive to look at what happens for small molecules where
there is often no solvent to worry about. They are often refined
using SHELXL, which does indeed print out the weighted R-value based
on intensities (wR2), the conventional unweighted R-value R1 (based
on F) and sigmaI/I, which
Oh cynic!
Eleanor
On 10/27/2010 09:01 PM, Simon Kolstoe wrote:
Surely the best model is the one that the referees for your paper are
happy with?
I have found referees to impose seemingly random and arbitrary standards
that sometime require a lot of effort to comply with but result in
little
I do not know if that's really cynical: I've had the case of a referee
recommending manuscript rejection because the title of the manuscript
was not appropriate. The editor followed the advice of the referee. A
proper refereeing job would have been to suggest that the authors change
the title
Dear all,
we have a his-tagged protein that shows a minor accompanying band in
SDS-PAGE, just above the main band. According to all other methods
available to us the material is homogeneous, the protein has the
correct
mass in MALDI-TOF, epitopes are recognized, etc. etc.
I know that the
Dear Peter,
it seems to me that you are having trouble with f2mtz and not with ctruncate, so
I am confused by the subject.
Can you please post
- the error message,
- the first couple of lines of the hkl-file you are trying to import (including
one or two reflections which are flagged for
Dear Sebastiaan,
isn't it the editor rather than the referees whom you have to convince? And if
the editor does not even understand how SDS-PAGE works and still considers
this a reason not to publish your article against your own expertise, maybe it
is worth changing the journal.
Finally, since
So I guess a consequence of what you say is that since in cases where there
is no solvent the R values are often better than the precision of the actual
measurements (never true with macromolecular crystals involving solvent),
perhaps our real problem might be modelling solvent?
Additional band on a gel might not be caused by his-tag. It is often a result
of different conformation/molecular shape and so the molecule travels with
different speed in the gel. We may wish for a homogenous sample (chemically and
structurally) but this is seldom true
See example:
Int J
Dear Crystallographers,
Thank you all for the emails. Below are some details of the procedures I
performed leading up to the problem.
The reflection file is my own data, processed in XDS and then flagging FreeR's
in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried
Hi Sebastian,
Under the assumption that the SDS in your assay does not completely unfold the
protein during electrophoresis (chemical impurity can be excluded because of MS
experiments, right?), how about adding some urea additionally to the SDS-PAGE
(or changing SDS concentration)?
GL Jan
It can sometimes be struggle to find the boundary between cynicism and
pragmatism!
I was, however, rather bemused by Dr Joosten's 7 rules of thumb -
probably all of which I use and have seen used by referees. Of course
I wouldn't want to blindly advocate any of them, however their use
Hello Peter,
I faintly rememeber a similar kind of problem, and think that if you replace
-1 with 0, the problem should go away. It seemed that -1 is not an allowed
flag for (some) ccp4 programs.
Please let us know if this resolves the issue.
Tim
On Thu, Oct 28, 2010 at 10:21:20AM -0400, Peter
In addition to bulk solvent, the other well recognized problem with
macromolecular structures is the inadequate description of disorder.
With small molecules, the Debye-Waller works much better because the
harmonic oscillator is indeed a good model there. Note that the problem
is not anisotropy
Not quite. I was trying to say that for good small molecule data, R1 is
usally significantly less than Rmerge, but never less than the precision
of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma
(or the very similar 0.5*Rpim).
George
Prof. George M. Sheldrick FRS
Dept. Structural
Hi Seb,
I'm not aware of the notion (and neither are your reviewers apparently) that a
His tag often results in two bands on a lane in SDS page. Why would that be?
Extra SDS binding to the positive patch?
Just wondering if there's any truth to your statement.
Also, since in this case there
Hi Tim,
sorry for my late reply - I just came back to the lab.
In the Babinet bulk solvent correction, no bulk solvent phases are used,
it is entirely based on amplitudes and strictly only valid if the phases
of the bulk solvent are opposite to the ones of the protein. And as
Sasha
Please let me clarify that it is by no means my intention to be rude to
any referees, nor to round up alternative explanations for the extra
band. The only thing I am after is a proper reference for the
phenomenon of his-tagged proteins producing an extra band at slightly
higher apparent molecular
Dear Sebastian,
Having personally purified upwards of 500 (I lost count really) of
His-tagged proteins, I can't say that I have the same awareness as you with
respect to the additional band being 'very common'. Depending on the kind of
expression system, size of your protein, conditions of
THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN
June 14, 2002 The Journal of Biological Chemistry, 277, 21397-21404.
His-tagged Iron cluster that runs as a doublet. Mass-spec show they are the
same species. They concluded that the protein binds SDS in two
So I guess there is never a case in crystallography in which our
models predict the data to within the errors of data collection? I
guess the situation might be similar to fitting a Michaelis-Menten
curve, in which the fitted line often misses the error bars of the
individual points, but gets the
Hello Tim,
Thank you for the suggestion. I have now tagged the working set as 1 and test
set as 0. Unfortunately, it still gives the same error about all Rfree being
the same, and only in c-truncate but not old-truncate. Perhaps I should install
6.1.3 and see if the problem still persist.
Why are you running [c]truncate? this is used to convert I - F and I would be
surprised if it recognised or preserved a FreeR column
Phil
On 28 Oct 2010, at 17:48, Peter Chan wrote:
Hello Tim,
Thank you for the suggestion. I have now tagged the working set as 1 and
test set as 0.
2) when I submit this to referees will they think my structure is
appropriate to draw these conclusions?.
Particularly question 2) can rarely be answered w/o looking at electron
density. Binding site details, ligand geometry, all have practically no
effect on the Rules of Thumb.
It is whilst
It is important to remember that if you have Gaussian-distributed errors and
you plot error bars between +1 sigma and -1 sigma (where sigma is the rms
error), then you expect the right curve to miss the error bars about 30%
of the time. This is just a property of the Gaussian distribution: you
The GUI task has the option to run (c)truncate after f2mtz (if you have
intensities in the input hkl file), and then uniqueify after that.
I can reproduce this problem. ctruncate is losing the freeR column. At
the moment, I don't know if this is a bug or a feature.
As a work around, you can run
Dear BB Sages,
I have a problem where I think I could very easily do the wrong thing.
And I don't really want to do that...
We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU -
Shelx, RESOLVE, ARPwARP. Cool.).
In p21 30 109 65 90 105 90 at 2.5A
However, we have now
Hi, all
I am trying to build an oligomer ligand. I have obtained all the cif files
for the monomers from HIC-UP. I tried to build several monomers in using coot.
Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC
1 Y ABC 2X-Y). However, when using
Hi Changyi,
I'm not sure if this will answer your question, but it is likely that the cif
file you are reading into Refmac is not in the correct format from Hic-Cup. I
would suggest reading your coordinate file for the ligand into the Dundee
server which will output a cif formats for
Hi.
Thanks for all of the helpful advice. Below is a summary of the
suggestions, along with some things that I have tried and the results thus
far.
1) Make sure that the crystals are protein and not salt.
My crystals absorb Izit dye well and shooting some initial crystals did not
produce any
On Thu, 28 Oct 2010 16:56:42 +0200
Dirk Kostrewa kostr...@genzentrum.lmu.de wrote:
In the Babinet bulk solvent correction, no bulk solvent phases are
used, it is entirely based on amplitudes and strictly only valid if
the phases of the bulk solvent are opposite to the ones of the
protein.
There are many cases where people use a structure refined at high
resolution as a starting molecular replacement structure for a closely
related/same protein with a lower resolution data set and get
substantially better R statistics than you would expect for that
resolution. So one factor in
On 28/10/10 20:12, Changyi Xue wrote:
Hi, all
I am trying to build an oligomer ligand. I have obtained all the
cif files for the monomers from HIC-UP. I tried to build several
monomers in using coot. Then I modified the pdb file and stated the
link in the pdb head (LINKR X ABC 1 Y
So let's say I take a 0.6 Ang structure, artificially introduce noise into
corresponding Fobs to make the resolution go down to 2 Ang, and refine using
the 0.6 Ang model--do I actually get R's better than the artificially-inflated
sigmas? Or let's say I experimentally decrease I/sigma by
Yes, but even the high-resolution structures cannot explain THEIR data to
within experimental error. You can see this if you download the CIF file
for one of the highest-resolution structures there is: 2vb1 (triclinic
lysozyme at 0.6 A), which contains both I and FC:
Bart Hazes wrote
There are many cases where people use a structure refined at high
resolution as a starting molecular replacement structure for a closely
related/same protein with a lower resolution data set and get substantially
better R statistics than you would expect for that
Hi
As I see you want to use link between ligands. You need to create this link
description first. It can be done using JLigand that is available from:
www.ysbl.york.ac.uk/mxstat/
There are tutorials how to create ligands and links. It should help you to
create links
We are updating at the
You're second suggestion would be a good test because you are dealing
with data from the same crystal and can thus assume the structures are
identical (radiation damage excluded).
So, take a highly diffracting crystal and collect a short-exposure low
resolution data set and long exposure high
On 10-10-28 04:09 PM, Ethan Merritt wrote:
This I can answer based on experience. One can take the coordinates from a
structure
refined at near atomic resolution (~1.0A), including multiple conformations,
partial occupancy waters, etc, and use it to calculate R factors against a lower
Zbyszek,
Since you mention I/sigmaI in your PDF, do you mean I/sigmaI or
I/sigmaI?
Do you mean I/sigmaI (in whatever rendition you choose) for the averaged
unique reflections or the I/sigmaI for the observations?
Also since one can adjust sigmaI in your scalepack program through the use
of the
Lake Wobegon!!!
For those outside the US and/or otherwise not familiar with that small town,
check out: http://en.wikipedia.org/wiki/Lake_Wobegon
Lake Wobegon, where all the women are strong, all the men are good looking,
and all the children are above average
The best use of modern
Dear Friends:
One post-doctoral
http://www.riken.go.jp/engn/r-world/info/recruit/k101029_s_rsc.html
position to work on structural biology project is available immediately at
RIKEN SPring-8 Center, Harima Institute, Japan.
With regards,
Kumarevel
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