Hi Todd,

in general you can refine any selected part of your model using any combination of refinement strategies. The are a few exceptions, and Cartesian SA is one of them: it is applied to all atoms. It is in the list of things to address.

SA refinement is good at early stages of refinement to correct gross errors, but it may do more harm than good for a relatively good structure.

I think in your case, Rwork/Rfree = 0.2478/0.2849, you can safely continue without doing SA. Although what you can try is the new feature - the local real-space refinement ("fix_rotamers=true" option). This is very powerful tool for automatic correction of side chains (you need to have one of the latest PHENIX versions for this). More details here:

http://cci.lbl.gov/~afonine/rsr.pdf

Regarding the B-factors: yes, this seems logical - if some U atoms gets displaces out of their density peaks then the refinement will try to smear them out so they do not contribute to the Fcalc, and enormous increased B-factors is the way to do this. If you allow occupancy refinement, their occupancies will probably refine to zero or close.

I'm not sure I understood this: are these atoms still moving out of density even if you do refinement without using SA? If this is the case then I do not know why this happens and I would really like to know. If you send me the data and model I might be able to figure this out.

Let me now if you have any questions or problems!

Pavel.


On 4/7/10 1:47 PM, Green, Todd wrote:
fixing atoms

Hello all,

I am refining a structure which contains several uranyl sites. I know the atoms should be there based on peaks in anomalous difference fourier maps, peak heights are greater than 10 sigma. Likewise we have solved the apo-structure before with the same heavy atoms. If I perform simulated annealing, the heteroatoms get pushed as far as 8 angstroms away. The Asp(s) and Glu residues that coordinate the uranyl ion then just move into the region of the map where the U has vacated. The resulting fofc difference map has a huge peak(>11 sigma) where the U should be. I noticed that if I inflate the starting b-factor for the U, the positions will stay put(relatively). What is the best thing to do here? Can I fix their positions, so that this does not happen? How do I do it? I found a post in the archives that says i can use: refine.sites.individual="not element U". Will this work for the SA? I know it works during refinement. Anyway, the data is to 3 angstrom. Here is the result from some SA runs:

following simulated annealing:
no u, starting b-factor for all atoms 20:
Final R-work = 0.2502, R-free = 0.2899
with u starting b-factor 20:
Final R-work = 0.2771, R-free = 0.3094
with u inflated starting b-factor for U:
Final R-work = 0.2682, R-free = 0.2983
After further manual rebuilding and TLS refinement(with u):
Final R-work = 0.2478, R-free = 0.2849


Also, during further refinement(if I reposition the U(s)), some of the U(s) still get displaced and the b-factors get extremely high, some as high as 500. I think this is due to them being displaced though. Other datasets which I have refined with phenix don't exhibit this behavior. Could it have something to do with NCS restraints on the protein?

any suggests?

Thanks in advance-
Todd 


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