Dear Pramod,
 
in addition to what has been said already, a few comments from my side. From what you write, the first thing I would do is to check the basics:
-Is the protein that crystallized the protein you think it is? Plasmids do get mixed-up and sometimes a more abundant natural protein with about the same molecular weight as the protein of interest gets purified.
-How do the diffraction images look like: are there strong ice-rings, multiple diffraction patterns or other problems? Could there have been misindexing in one of the directions?
-Pointless is not the final word. Especially in cases with pseudo-crystallographic symmetry, Pointless may be led to believe that the space group has higher symmetry than the true space group. I would definitively also try molecular replacement with lower symmetry, e.g. P1 and P2x.
-Did you rebuild your structure before refinement? If some loops are in wrong density, the whole refinement may go in the wrong direction. The first maps after MR but before refinement are bad given the high Rfactors. However, they tend to be unbiased and may show how to rebuild. However, if the density is sort of ok without any strong (diffference) density suggesting how to rebuild, you have a problem. By the way, the pictures you showed looked contoured at a very low level. I would go to a higher contour level to find out which parts to trust, and which parts to delete.
 
Good luck!
Herman
 
PS: it never hurts trying to grow more crystals. Maybe another crystal does not have these problems.

Von: CCP4 bulletin board [mailto:[log in to unmask]] Im Auftrag von Pramod Kumar
Gesendet: Montag, 17. Juni 2013 23:29
An: [log in to unmask]
Betreff: Re: [ccp4bb] str solving problem

Dear Abhinav

 I would suggest you integrate in p1 and run pointless.
after integrating in p1 and running the pointless its still concluding the SG c2221.

How do you determine the resolution cut off of 2.9A?
the basis of resolution at 2.9A* was the completeness and I/Sigma value.

Two molecule in ASU further supported by Matthews coefficient and solvent percentage content.

Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw?
Have you trimmed loops and floppy regions in the model?

its not homology model, I have used chainsaw to make the model for molecular replacement,
and trimmed the model for floppy, loopy ambiguousness regions.

plz see the snap shot of the model.


thanks and regards...

pramod





On Tue, Jun 18, 2013 at 1:16 AM, Abhinav Kumar <[log in to unmask]> wrote:
Hi Pramod,
 I would suggest you integrate in p1 and run pointless.
If your Rfree is above 0.45, I wouldn't trust the model.

How do you determine the resolution cut off of 2.9A?

In MR, the score should improve when the program places the second copy to the first molecule.

Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw?
Have you trimmed loops and floppy regions in the model?


Thanks,
Abhinav

JCSG@SSRL, SLAC
(650) 926-2992

On 06/17/2013 12:36 PM, Pramod Kumar wrote:
Dear Abhinav Kumar
thanks for kind suggestions


I have tried as follow.


1. You should try to identify the correct space group first.

        *integration in p21 given the following statics in pointless

Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation

              [h,k,l]              0.566    0.957    0.094     25580      0.00
              [l,-k,h]             0.434    0.853    0.195     25580      0.20

        *integration in c2221 given the following statics in pointless

Spacegroup TotProb SysAbsProb Reindex Conditions



     ( 20)    0.997  0.997                         00l: l=2n (zone 1)
           moreover Zanuda and molrep's chekall space group option also suggests c2221,

2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success.Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser?

      * this is the limitation of my data that came from three month old crystal, there are two molecules in ASU (what is the extant to say "reasonably good" please ?), initially phaser was failed, with the balbes output as ensemble it worked with following profile ..

** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 ***

*************************************************************************************


   Current is Best Solution (first)
      New Best LLG : 101.7 (8.70)
      Best Search Component so far: ensemble1 


*************************************************************************************
*** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING             2.5.2 ***
*************************************************************************************


   Resolution of All Data (Number):        2.91  47.64 (15877)
   Resolution of Selected Data (Number):   2.91  47.64 (15877)




   Refinement Table (Unsorted)
   ---------------------------
   #+ = input number    #* = results number
   #+   #*  (Initial LLG & Rval) (Refined LLG & Rval) Unique   =# Tmplt SpaceGroup 
   1    1             908.3 48.8          1804.3 46.4    YES            C 2 2 21   




   Refinement Table (Sorted)
   -------------------------
   #+ = input number    #* = results number
   #+   #*  (Initial LLG & Rval) (Refined LLG & Rval) Unique   =# Tmplt SpaceGroup 
   1    1             908.3 48.8          1804.3 46.4    YES            C 2 2 21   


but again the refmac  is showing

R factor 0.4404 0.4203

R free 0.4955 0.5027

Rms BondLength 0.0254 0.0093

Rms BondAngle 3.1234 1.4581

Rms ChirVolume 0.1741 0.0706









3. Did you check for twinning?

TWINNING SUMMARY


Twinning fraction from H-test: 0.00

L-statistic from L-Test: 0.48


Relation between L statistics and twinning fraction:

Twinning fraction = 0.000 L statistics = 0.500:

Twinning fraction = 0.100 L statistics = 0.440:

Twinning fraction = 0.500 L statistics = 0.375:

NO Twinning detected
thanks and regards

pramod...



On Mon, Jun 17, 2013 at 10:15 PM, Abhinav Kumar <[log in to unmask]> wrote:
Hi Pramod,

1. You should try to identify the correct space group first. Did you integrate in p1 and run pointless?
2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success. Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser?
3. Did you check for twinning?
Thanks,
Abhinav

JCSG@SSRL, SLAC
(650) 926-2992

On 06/17/2013 09:35 AM, Pramod Kumar wrote:
Dear group

I have a crystal data diffracted  around 2.9 A*,
during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO, now the model for molecular replacement with closest identity of 31 given a contrast of 2, score 0.30 and wrfac 0.60. but "balbes" uses different models with lesser identity,

no matter which way I am going the rFree keep on increasing during refinement in refmac, when I build the model in coot with deletion and addition of residue it starts with relatively low but gradually rises through almost all cycles although model fits to the density well and residue are  building, coot validation parameters are also reasonable OK for geometry, rotamer, density fit,..

now my question....

* where should i first check for possible correction?

* In molecular replacement what should be the red line for identity and related criteria?

* if initial rFree starts around 50, how likely that its not the right way?

* my rms bond angle is close to 1 while the bond length is 0.01 and chiral 0.1 concludes what is serious?



sincere apology for amateur query if any...

thanks in advance


pramod



--
************************************************
Pramod Kumar.
Graduate Student.
Crystallography lab.
Department Of Biotechnology.
Indian Institute Of Technology Roorkee
Uttranchal.247667 
India
+919359189657.
************************************************






--
************************************************
Pramod Kumar.
Graduate Student.
Crystallography lab.
Department Of Biotechnology.
Indian Institute Of Technology Roorkee
Uttranchal.247667 
India
+919359189657.
************************************************