pramodthanks and regards...plz see the snap shot of the model.and trimmed the model for floppy, loopy ambiguousness regions.its not homology model, I have used chainsaw to make the model for molecular replacement,Two molecule in ASU further supported by Matthews coefficient and solvent percentage content.the basis of resolution at 2.9A* was the completeness and I/Sigma value.after integrating in p1 and running the pointless its still concluding the SG c2221.Dear Abhinav
I would suggest you integrate in p1 and run pointless.
How do you determine the resolution cut off of 2.9A?
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?
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,
AbhinavJCSG@SSRL, SLAC
(650) 926-2992
On 06/17/2013 12:36 PM, Pramod Kumar wrote:
pramod...Dear Abhinav Kumar
thanks for kind suggestions
* 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 ..
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?
** 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
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,
AbhinavJCSG@SSRL, SLAC
(650) 926-2992
On 06/17/2013 09:35 AM, Pramod Kumar wrote:
Dear groupI 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.
************************************************