> Date: Mon, 21 Nov 2011 12:27:04 +0000
> From:
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[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
>
