[ccp4bb] [ccp4]questions about SHELXD
Hi all, I was trying to use SHELXD program for protein peptides (6-7 residues) for the very first time, and I got the .pdb file which should be the correct solution. However, in the .pdb file, the atoms are labeled as ABC and they are not recognized as amino acids. My question is normally what program can I run after SHELXD, to put those atoms into residues in the correct order so that I can use refmac to refine the structure? Another question is, I have another peptide which has P1 space group, and the SHELXD won't start and it said the cell is too small to put atoms randomly, in this case, can I still use SHELXD for the structure solving and what should I do? If not, what other programs can I use to solve small peptide structures with 6-7 residues? Thanks for your help!! Lu
[ccp4bb] a question for PDB_extract for RCSB depositation
Dear All, For the PDB_extract, one item to be filled is Deposit Structure Factor Used for Final Refinement. If this way the structure factor which could be downloaded from RCSB after depositation will be not the final structure factor corresponding to the final deposited PDB file, as for what we deposited is Structure Factor Used for Final Refinement, rather than the Structure Factor after Final Refinement. I am looking forward to getting a reply from you on whether I am right or not. Cheers, Dialing
Re: [ccp4bb] Extra positive density seen after TLS refinement?
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.auwrote: 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
[ccp4bb] question about input .hkl file for SHELXD
Hi, I was confused with the input .hkl file for SHELXD. I was using ccp4 to prepare these files, and I am not sure whether I was doing it correctly. 1st I did scale2mtz to generate .mtz file 2nd I used mtz2various to convert .mtz to .hkl format. I also found a program prephadata which can convert .mtz to .hkl, however, the hkl file generated from these 2 programs are different from each other. I don't know which one to use. I was wondering whether anyone of you have tried to do the conversion, and is there any special option box from the ccp4 that I need to click when I used the scale2mtz and mtz2various? Thanks for your help! Lu
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
