Dear Shiva,
First a word of caution. Your diffraction pattern is the convolution of the molecular transform and the lattice transform. Your molecular transform (determined by the orientation of your model) will almost certainly be right. The influence of e.g. one misidentified screw axis will not be very strong. This means that if you leave out a strand, density may come back, even with a misidentified screw axis. I would reprocess the data in P1 and generate a model (2 molecules in 1 pdb file) consisting of one molecule at (0,0,0) and one at (0,0.5,0.5) in the orientation you found already and run molrep with that model.
It could also be illuminating to look at what is behind the off-origin peak: it means that you have one molecule (say A) at (0.0.0) and one molecule (say B) at (0,0.5,0.5) which are very similar, but not identical. If they were identical, your off-origin peak would be crystallographic and an origin peak. If you look at the relative phases, the phase of molecule A would be 2pi(h*0+k*0+l*0)=0 whereas the relative phase of molecule B would be 2pi(h*0+0.5*k+0.5*l)=pi(k+l).
The total diffraction is then: Ftotal = FA (cos(0) + isin(0)) + FB (cos(pi(k+l) + isin(k+l)).
For k+l even pi(k+l)= 0 or 2pi and Ftotal = FA (1 + 0) + FB ( 1 + 0) = FA + FB (strong reflections)
For k+l odd pi(k+l)= pi and Ftotal = FA (1 + 0) + FB (-1 + 0) = FA - FB (weak reflections, zero if FA=FB)
This means, you could also discard all k+l reflections by reprocessing the data in the smaller unit cell and run molrep with that data. You will have to search for only one molecule and the final density will be the sum of molecules A and B. This may give you hints how the packing of your larger unit cell may look like. As I said before, these almost crystallographic NCS is extremely trickey and can easily fool automatic programs and crystallographers alike.
Good luck!
Herman
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Shiva Kumar
Sent: Thursday, March 22, 2012 11:29 PM
To: [log in to unmask]
Subject: [ccp4bb] Refinement with pseudo-translation
Dear CCP4bb members
I have a 3.0 Å dataset which has an off-origin peak of height 36% in patterson map. The peak is at fractional co-ordinates 0, 0.5, 0.5. Data has been indexed in P2(1)22(1) SG using HKL2000. I have located all the molecules in asu (as far as I know) using Molrep with the 'locked rotation' and 'Use' PST feature. After 1 round (20 cycles) of rigid body and 1 round (10 cycles) of restrained refinement (Refmac), the R and Rfree are 49 and 53 %. Although the R factors are very high, I feel the solution might be correct because the electron density follows c-alpha trace in almost all places. To be sure, I deleted a beta strand from the structure's core and repeated the refinement and found that the electron density for the strand was still present (I have no experimental phases). I have the following questions:
1) Are the final R factors never expected to reduce to acceptable values given the 36% off-origin peak?
2) What is the best way to settle the SG? I was considering P2(1)2(1)2(1), P2(1)22, P2(1)2(1)2 and P2(1)22(1), considering the off-origin peak is at 0,0.5,0.5. I found all the molecules in asu only using the P2(1)22(1) data in Molrep. Is this the best way to settle my SG?
3) Some CCP4bb archives advise either refining against weak and strong reflections alternatively (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1010&L=ccp4bb&D=0&1=ccp4bb&9=A&J=on&d=No+Match%3BMatch%3BMatches&z=4&P=204490) or refining against medium intensity reflections. Should I also be doing these things? If yes, then what is the best way of doing it?
Your suggestions and corrections to my interpretation of our data would be appreciated.
Regards
Shiva
|