MolProbability score doesn't mean too much in your case, since you are essentially using a 1.5 A model against a 3 A database. The differences in the blobs might caused by the different delta sigma settings when you were viewing these two models. I have successfully used TLS for a 3 A dataset before. The blobs mean the discrepancies between you model and your data, no matter you are using TLS or not. If TLS doesn't give you better statics and map density, I would leave it out or change the TLS setting. I wouldn't put any molecule in the densities you provided, as the model looks already congested enough from the angle I see. I might be wrong w/o the information of map setting.
Best, Nian On Sat, Feb 18, 2012 at 7:36 PM, Naveed A Nadvi <nnad2...@uni.sydney.edu.au>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. My questions are: > > > > 1) Is TLS suitable for our dataset (3 A)? > > 2) Is TLS refinement independent of NCS refinement or should I define my > NCS based on the 8 TLS groups? > > 3) Is it normal to see extra positive density after TLS refinement and > what does it mean? > > 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 > >
