Dear Naveed A Nadvi
I think that your results highlight the fact that modelling the
disorder/complex ordering of your crystal is relevant and in general,
that we should take care in optimizing B factor refinement as a strong
factor for model improvement.
In this sense I would not relay in one TLS group definition, even
though you have obtain better results comparing no TLS vs TLS. Try
common sense definitions also: does the protein have a hinge between
two domains, use the domains as TLS groups, etc. Then, look for
optimal NCS between TLS groups also.
Best wishes
Horacio
Quoting Naveed Ahmed Nadvi <nnad2...@uni.sydney.edu.au>:
Dear Crystallographers,
Thank you for your responses. The density map levels were 0.11e/A^3
(3.00 A) for both images with and without TLS refinement. When I
superposed the deposited structure I could see the extra density was
due to water moleucles that were seen in the higher resolution
deposited structure. It is so interesting how I could not see them
in my data without doing TLS.
Performing the TLS refinement improved overall parameters:
no TLS/TLS
R 0.2425/0.2334
R-free 0.2809/0.2748
RMS BondLen 0.0092/0.0074
RMS BondAngle 1.1812/1.1407
ChirVol 0.0806/0.0779
My question is, do the positive density seen after TLS refinement
justify adding these solvent molecules especially when they were not
observed without TLS refinement?
Thank you once again for your insights!
Naveed
________________________________________
From: Ethan Merritt [merr...@u.washington.edu]
Sent: Sunday, 19 February 2012 3:10 PM
To: Naveed Ahmed Nadvi
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Extra positive density seen after TLS refinement?
On Saturday, 18 February 2012, Naveed A Nadvi wrote:
Dear crystallographers,
I am fairly new in crystallographic work and structure
determination, but I thought this would be the best place to post
my questions. We had collected structural data for a protein that
diffracted to 3 A. We had used a previously deposited structure
(1.5 A) for molecular replacement. Our final structure used NCS
restraints refinement over 4 chains within the assymetric unit. We
were able to assign some water moleules using COOT and subsequently
removed 'bad waters' manually. We used automated settings when
dealing with these water molecules. In all cases these water
molecules were found in the same positions as the initial structure
(1.5 A) that we had used as a search model. This gave us
confidence in the placement of our water molecules. Finally we had
run validation tools (MolProbity) and our structure was found to
be with Molprobity score within the 100th percentile.
We then performed a TLS refinement (from TLSMD) to further improve
R values. We used the final MolProbity-validated structure using 8
TLS groups and using PureTLS with constant B factor (50). We are
observing large positive densities from the subsequent REFMAC5
refinement that are otherwise not observed in the absence of TLS
refinement.
Is it possible that the peaks are not higher in terms of absolute
electron level,
but only in terms of RMSD? That is, if the TLS treatment cleans up the map
everywhere, then whatever peaks are left will deviate more from the
(now lower)
mean value even though their absolute size is the same.
In other words, the "3 sigma" contours in your first map may be more like
6 sigma contours in your second (cleaner) map.
My questions are:
1) Is TLS suitable for our dataset (3 A)?
There is no universal answer to that. You just have to test for
yourself each time.
Certainly TLS can help a lot at 3A for some structures. In general the more
anisotropy is present, the more it helps to include it in your model
somehow -
and TLS is a "cheap" way to include it in your model. But if your
structure does
not have much anisotropy, then adding TLS to describe it won't have
much effect.
2) Is TLS refinement independent of NCS refinement or should I
define my NCS based on the 8 TLS groups?
They are not the same thing at all.
3) Is it normal to see extra positive density after TLS refinement
and what does it mean?
See possible explanation above.
Ethan
4) We had PEG4000 and Tris in our crystallization buffer. Could
these 'blobs' represent these molecules or short water chains? I
have attached images of the largest blob.
Any comments and suggestions would be highly appreciated.
Kind regards,
Naveed A Nadvi
Faculty of Pharmacy,
University of Sydney, Australia
