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: c...@ysbl.york.ac.uk
> To: ccp4...@hotmail.com
> CC: CCP4BB@JISCMAIL.AC.UK
> 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
> >
>
