Dear Prof Dodson, I agree with you. Instead of further refining mutant structure, right now I am looking in to Apo structure. Which is at 2.74A and has R/freeR 22.7/27.9. But I have a molprobity profile like this Clashscore, all atoms: 33.73 50th percentile* (N=185, 2.740Å ± 0.25Å)Geometry Poor rotamers 8.11% Goal: <1%Ramachandran outliers 0.52% Goal: <0.2%Ramachandran favored 95.19% Goal: >98%Cβ deviations >0.25Å 2 Goal: 0MolProbity score^ 3.04 53rd percentile* (N=5278, 2.740Å ± 0.25Å)Residues with bad bonds: 0.00% Goal: 0%Residues with bad angles: 0.34% Goal: <0.1% If i fix the all the possible outliers and refine the structure it wouldn't improve the molprobity scores. I am not happy about the above scores at all. I am wondering if this is an indication of any wrong in the structure or is this common for an enzyme. I have reprocessed the data to make sure space group is same C2221.Any help would help me understand this and learn more. ThanksRaj > Date: Mon, 21 Nov 2011 12:27:04 +0000 > From: [log in to unmask] > To: [log in to unmask] > CC: [log in to unmask] > Subject: Re: [ccp4bb] help with the structures > > I think you are proving yet again that refinement at 3.3A is not easy. > Indeed there are probably multiple conformations for parts of the > structure and that may well be why your data is at low resolution and > anisotropic. Maybe this is the best you can do.. > > I think I would make sure the apo structure is as good as it can be, > then fit that to the 3.3A data set, and only use that 3.3A data to > deduce whatever features differ from the APO structure. > > Eleanor > On 11/19/2011 12:09 PM, Rajesh kumar wrote: > > > > Dear All, > > > > > > > > We have an anisotropic dataset of 3.3 A and it was solved (not by me) with P6522 with R/freeR > > 29.1/37.3. > > > > > > > > I got the corrected > > mtz file by plugging in the .HKL (P6122) file to anisotropy diffraction server at > > 2.04 A. I reindexed this p6122 to p6522 and extended the resolution and refined > > (refmac) the structure to R/freeR > > 36.40/38.50. With aotoncs option, fixing all Ramachnadran and rotamer > > outliers I got it 30/32. When I added waters and it went down to 27.5/31.2. At > > this point I recognized that my new .mtz file from anisotropy server has > > different R flag than the earlier one (3.3A) so I copied the R flag and did refinemnt to get R/Rfree 0.2682/0.3247. When I looked at > > the refined structure I found more outliers > > than I fixed in earlier round. I did fix all the outliers and without NCS and > > waters it gives R/Rfree 0.2906/0.3325. At all the stages I look at outliers at > > molprobity server which suggested structure is 10th percentile and after > > refinement more outliers comes back. At stage-1 map looked far better so was > > happy that anisotropy correction has worked for me (this was my first time > > handling this type of dataset) but further refinement didn’t make it look any > > better.I use both refmac and autoBuster for refinement. http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048657095/ > > This protein is an human enzyme and a bacterial homologue > > which has 38% identity has been used to solve the Apo structure (2.7 A, pC2221, R/freeR > > 23.03/27.96, molprobity is around 50th percentile). I looked in to this I try to fix all the outliers and try to improve > > molprobity score but it just refused to improve as after refinement I get more > > outliers. This Apo structure was used to solve the mutant structure at > > 3.3 A. I believe that both structure could > > have better R/freeR and excellent molprobity scores than what they have now. I am not able to recognise > > if there is any problem in Apo structure and if errors have come to mutant so > > both of them refuse to improve. > > > > > > I wondered if there is any model bias (I don’t know if it’s > > the case but nothing was coming to my mind) so thought using ARP/wARP classic > > to build model from existing model but it complained that "The wilson plot > > is very bad and ARp/wARP is very unlikely to run in a sensible way. Please > > check your data" . http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048687955/ > > > > > > > > At this point I dont know how to systematically dissect this problem. I know there could be wrong in several places but with my only '2-3 structure experience' I am not able to identify the regions to look for > > error but I think something is not right. I really appreciate if you give me > > some suggestions/ideas/directions/tips so that I could recognize problem and > > improve structure and learn some more. > > I appreciate your valuable time. > > Regards,Rajesh >