SAVE THE DATE! The first ever SBGrid user summit 'Quo Vadis Structural
Biology?' will take place on May 5th and 6th 2008 in Boston, Massachusetts.
We will have a number of talks and workshops focused on structural biology
computing.
This two-day conference will include lectures and hands-on compu
At least that's been our experience.
Andy
(No affiliation with NEB)
Hi, everyone:
Just to give both sides to this story, though, we've had phenomenally bad luck
with the NEB Intein system (tagged on either the C- or N- terminus). The
protein expressed and purified beautifully, and post-cleavag
Ooh..la la! Where were you 12 hrs ago when we were suffering brain damage!
Cheers!
Raji
PS: Thanks!
>Oh dear - too late :-(. You can do it in Coot too! (The solution is on
>the Coot Wiki now)
>
>http://xanana.ucsc.edu/~wgscott/xtal/wiki/index.php/Coot#Example_Scheme_Script_7:_Applying_arbitrar
Raji Edayathumangalam wrote:
Thanks everyone for all your suggestions.
What's the issue? Each residue has one CSV value (per residue value) and the PDB line contains many
lines of B-values per residue. This would leave us with no easy one-to-one line correlation between
the B-value column and
If you think about it, there is an analogy to relaxing geometrical
constraints, which also allows the refinement to put atoms into
"density". The reason it usually doesn't help Rfree is that the density
is spurious. At least some of the incorrect structure determinations of
the early 90's (that
Thanks everyone for all your suggestions.
What's the issue? Each residue has one CSV value (per residue value) and the
PDB line contains many
lines of B-values per residue. This would leave us with no easy one-to-one line
correlation between
the B-value column and the CSV column. From what I u
Hi Raji,
I think phenix.pdbtools can do it (if I correctly understood what you
want to do):
http://www.phenix-online.org/documentation/pdbtools.htm
Example:
phenix.pdbtools model.pdb set_b_iso=25.3 selection="chain A and resname
ALA and name CA"
this will set all B=25 for all CA atoms in
Hi People,
I post this on behalf of my colleague. My colleague has a file containing
chemical-shift values for
the 150 aa in his structure. He also has the PDB file for the crystal
structure. Now, he would like
to replace the B-factor column with the CS values to make some figures.
It would be
Hi Phil,
Here I will disagree. R-free rewards you for putting in atom in density
which an atom belongs in. It doesn't necessarily reward you for putting
the *right* atom in that density, but it does become difficult to do
that under normal circumstances unless you have approximately the righ
If circumstances require a C-terminal tag, the intein system from New
England Biolabs has worked very well for us. The pTYB1 plasmid encodes a
fusion between your protein of interest, a viral intein (think "protein
intron") and a chitin binding domain. The fusion adsorbs to a chitin resin
and all
Hiya
We usually add all N-terminal tags with a TeV cleavable linker. C-terminal
tags always seem a pain to remove cleanly, because most highly specific
recognition sequences (such as TeV) take advantage of P rather than P'
specificity so you are usually left with a five (or so) residue tail.
Hello,
I'd appreciate it if anyone could provide information (experiences or
publications) on the following:
1. Put a tag such as FLAG, V5, etc etc, at the N- and/or C-termini, in order
for specific detection, but not interfering with protein folding/structure;
2. Is a linker between the tag and
Dean Madden wrote:
Hi Ed,
This is an intriguing argument, but I know (having caught such a case as
a reviewer) that even in cases of low NCS symmetry, Rfree can be
significantly biased. I think the reason is that the discrepancy between
pairs of NCS-related reflections (i.e. Fo-Fo') is genera
Agreed, and this is even more true if you consider R-merge is calculated
on I's and Rfree on F's, Rmerge of 5% should contribute 2.5% to Rfree;
and furthermore errors add vectorially so it would be
more like ,025/sqrt(2).
I guess I have to take all those other errors that have to do with
the inab
Hi Ed,
This is an intriguing argument, but I know (having caught such a case as
a reviewer) that even in cases of low NCS symmetry, Rfree can be
significantly biased. I think the reason is that the discrepancy between
pairs of NCS-related reflections (i.e. Fo-Fo') is generally
significantly s
Here I will disagree. R-free rewards you for putting in atom in density
which an atom belongs in. It doesn't necessarily reward you for putting
the *right* atom in that density, but it does become difficult to do
that under normal circumstances unless you have approximately the right
structur
Dear Ed,
I don't see how you "decouple" symmetry mates in the case of a wrong
space group. Symmetry mates should agree with each other typically
within "R_sym" or "R_merge" percent, eg; about 2-5% . Observed and
calculated reflections agree within "R_Factor" of each other, so about
20-30%. Th
It is true that multicopy refinement was essential for the suppression
of Rwork. However, the whole point of the Rfree is that it is supposed
to be independent of the number of parameters you're refining. Simply
throwing multiple copies of the model into the refinement shouldn't have
affected R
Actually the bottom lines below were my argument in the case
that you DO apply strict NCS (although the argument runs into
some questionable points if you follow it out).
In the case that you DO NOT apply NCS, there is a second
decoupling mechanism:
Not only the error in Fo may be opposite for th
While NCS probably played a role in the first crystal form of MsbA (P1,
8 monomers), this is also the one that showed the greatest improvement
in R-free once the structure was correctly redetermined (7% or 14%
depending on which refinement protocols you compare).
The other crystal form of MsbA
One small step for a crystallographer, one giant leap for mankind! (*)
--
For those of you who didn't see it, the following was posted to the PDB
mailing list last week:
Announcement: Experimental Data Will Be Required for De
Dean Madden wrote:
Hi Dirk,
I disagree with your final sentence. Even if you don't apply NCS
restraints/constraints during refinement, there is a serious risk of NCS
"contaminating" your Rfree. Consider the limiting case in which the
"NCS" is produced simply by working in an artificially low
A few comments that you might find useful:
1. yes, even if you don't apply NCS restraints/constraints there will
be correlations between
reflections in cases of NCS symmetry or pseudo-crystallographic NCS
symmetry.
2. Fabiola, Chapman, et al., published a very nice paper on the topic
in A
Bottom line: thin shells are not a perfect solution, but if NCS is
present, choosing the free set randomly is *never* a better choice,
and almost always significantly worse.
hmmm ... I wonder if that is true. For low order NCS (two- three-
fold, even five-fold) I don't believe that thin she
Hi Dirk,
I disagree with your final sentence. Even if you don't apply NCS
restraints/constraints during refinement, there is a serious risk of NCS
"contaminating" your Rfree. Consider the limiting case in which the
"NCS" is produced simply by working in an artificially low symmetry
space-grou
Dear Crystallographers,
The recent conversation about NCS got me thinking about something I have been
wondering about for a while.
Imagine a structure with twenty-fold NCS which diffracts to 2A versus a no-NCS
structure of the same resolution--obviously the first model will be better than
the
Dear Yang,
I have a protein which has the function unit as a dimer. I got two
structures of it. One is the native structure, one is the mutant
structure. Both structures are dimer in ASU. In the native
structure the two chain look the same, but in mutant structure one
chain still keep
Dear CCP4ers,
I'm not convinced, that thin shells are sufficient: I think, in
principle, one should omit thick shells (greater than the diameter of
the G-function of the molecule/assembly that is used to describe NCS-
interactions in reciprocal space), and use the inner thin layer of
these
Dear Kay,
That looks to be an excellent start. I should like to add SHELXL as well
as SHELXC/D/E, there are regularly questions about it in CCP4bb. I'm not
quite clear about how to generate enough material to make it useful,
obviously you don't want to simply copy the SHELX manual, and similar
When we develop our gel we recently keep getting a horizontal line of
stain at 5 mol weight in all lanes of our gel. (This is not a
feature of interest except that the protein that we are interested happens
to be 5 mol weight). I would appreciate any ideas on how we can get
rid of
It is important when using NCS that the Rfree reflections be selected is
distributed thin resolution shells. That way application of NCS should not
mix Rwork and Rfree sets. Normal random selection or Rfree + NCS
(especially 4x or higher) will drive Rfree down unfairly.
Doug Ohlendorf
-Origi
Dear all,
I want to bring two crystallographic wikis to your attention, asking for
your contribution:
1) CCP4 user community wiki:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page
This wiki was planned by Tassos and me, and Artem and Clemens joined us
lately. Unfortuna
Hi Mike
Sometimes keratin from your skin (fingers, nails, etc) appear in the gels
as a band around 48-52 kDa. Take care in the handling of the
electrophoresis should be a solution.
Other possible reason could be a contamination of the sample loading
buffer, also with keratin or with other protein
Hello,
When we develop our gel we recently keep getting a horizontal line of stain
at 5 mol weight in all lanes of our gel. (This is not a feature of
interest except that the protein that we are interested happens to be 5
mol weight). I would appreciate any ideas on how we can get rid o
Forwarded on behalf of Lachlan Cranswick.
--dvd
-- Forwarded message --
2008 Kyoto IUCr Crystallographic Computing School - Sharing our knowledge
(preliminary announcement)
Kyoto Crystallographic Computing School
Kansai Seminar House, K
Dear All,
I have a protein which has the function unit as a dimer. I got two
structures of it. One is the native structure, one is the mutant
structure. Both structures are dimer in ASU. In the native structure the
two chain look the same, but in mutant structure one chain still keep the
sa
Hello Ainsley
I'm not sure what the MOLREPLIB is used for, I checked the Molrep source
code and references to it are commented out. So it seems most likely
that this is now just an historical curiosity, and can be safely ignored.
I suspect that your question regarding Phaser has been lost in the
37 matches
Mail list logo
