Hi all,

thanks for all your help so far, and as we ended up in a more general discussion about temperature factor refinement at not-so-great resolution, here is a quick summary of what I'll try out:

1.) Refine overall B's instead of isotropic B's.

2.) Use isotropic B's with the following (combinable) options:

a) Isotropic in the beginning, grouped B's in the end.
b) Use tight geometric restraints (I'm doing this anyways).
c) Use tight B restraints rather than grouped CNS B's (not geometrically restrained, and most likely not restrained by NCS).

3.) Use not so tight B restraints, but tight geometric restraints with individual or grouped B's, plus TLSMD server and multi-group TLS refinement.

4.) Use CNS and try Mark White's tools, and simulated annealing.

5.) Use phenix with weighted nearest-neighbor restraint.

...and some remarks:
* Of course I never wanted not to refine any B factors! I just wanted to see their contribution/influence on the refinement and explain their strange behaviour.
* Luckily, I have NCS :o) Thanks for your good wishes, Mark.


Eva

2007/4/18, Eva Kirchner <[log in to unmask]>:

Hi,

I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%.

Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac "flattens" them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution.

Here is what I tried further:

* I de-selected "Refine isotropic temperature factors" in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?)

* I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC <wbskal><sigb1><sigb2><sigb3><sigb4>), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even "flatter"). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs.

Thanks for any thoughts on this,

Eva