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CCP4bb <-- 2007 <-- April 2007 <-- 19 April 2007
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Subject: Re: Protein expression in Minimal media (M9)
From: "Richard P {- dot -} Grant" rpg {- at -} USYD {- dot -} EDU {- dot -} AU
Date: 2007-04-18 		Next message:
Subject: Re: Protein expression in Minimal media (M9)
From: "John A {- dot -} Newitt" newittja {- at -} COMPUSERVE {- dot -} COM
Date: 2007-04-19


Subject: Summary - Stop Refmac from refining B factors?
From: Eva Kirchner eva {- dot -} kirchner {- at -} UNI-TUEBINGEN {- dot -} DE
Date: 2007-04-19
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 :
>
> 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
> ), 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
>
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