Dear Eva and Harry and others,
I am not sure that an overall B-factor is the best solution for a 3.2 Å
structure. In general, the true B-factors will vary a lot for different parts
of the protein and poor diffracting proteins often have parts which are
partially or completely disordered. An overall B-factor is a very poor estimate
of such a true B-factor distribution and together with the low-resolution data
will create a lot of model bias i.e. very nice density for side chains which
are, in fact, disordered.
I have struggled a long time with a 3.2 Å antithrombin data set and as luck
would have it, the most interesting and unknown part of the structure was a
loop with extremely high temperature factors. All standard protocols I tried
produced very nice density for the model I put in, but did not tell me anything
of how to build the unknown parts.
A side remark by Randy Read finally helped me to solve this problem, which was
to reduce model bias by using very tight geometric restraints (e.g. not allow
the xyz positions produce bias) and individual temperature factors. What I
oberved was that wrongly fitted residues would be pushed out of the density due
to the tight geometric restraints, the artificially good electron density of
some flexible parts would get weaker and most importantly: electron density
appeared were I could fit the important reactive site loop. It worked in this
case, it may not work in other cases and at 3.2 Å one should always proceed
very cautiously and try to use whatever information (biochemical, mutations) is
available to validate the model.
This was my experience and I have never used an overall B-factor afterwards.
Herman Schreuder
________________________________
Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Harry
M. Greenblatt
Gesendet: Mittwoch, 18. April 2007 14:38
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Stop Refmac from refining B factors?
BS"D
Dear Eva,
You should be refining an overall temperature factor at that
resolution. It's one of the choices in the list, instead of "isotropic".
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
-------------------------------------------------------------------------
Harry M. Greenblatt
Staff Scientist
Dept of Structural Biology [EMAIL PROTECTED]
Weizmann Institute of Science Phone: 972-8-934-3625
Rehovot, 76100 Facsimile: 972-8-934-4159
Israel
