Re: [ccp4bb] Stop Refmac from refining B factors?

[Mark A. White]

Eva,

I have used CNS to refine several low resolution structures.  I have
even developed several tools specifically for low-resolution refinement
in CNS (see http://xray.utmb.edu/PMB/index.html#BPATCH).  However, I
agree with the advice given by most of my colleagues, refine isotropic
B-factors in REFMAC, but do not forget to use TLS.  My earlier post (see
below) bounced, due to my having multiple email addresses, not all of
which are privileged to post to CCP4BB.  Both CNS and PHENIX have some
nice features, not available in REFMAC, such as composit-omit maps and
simulated annealing, which really reduce the model bias.  So I often
switch back and forth between CNS and REFMAC.


On Wed, 2007-04-18 at 15:51 +0200, [EMAIL PROTECTED]
wrote: 

> 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 agree, but the problem is that the traditional isotropic B-factor
refinement is not restrained properly.  Many years ago Ian Tickle
determined that the correct restraint for isotropic B-factors had a B**2
dependence.  I have continued his analysis, and have found a general
restraint weight that seems to work for any resolution data.  So far I
have refined isotropic B-factors at 4 A, with a noticeable decrease in
free-R (http://xray.utmb.edu/PMB/index.html#BPATCH), over a multiple
grouped-B approach.  This improved B-factor restraint patch is available
with PMB (http://xray.utmb.edu/PMB), for use with CNS 1.1.  Alternately,
the isotropic B-factor restraints in PHENIX uses a weighted
nearest-neighbor restraint which should be robust, and may possibly work
at low resolution also.

Another work-around is to apply very tight restraints in terms of target
values.  Your bond rmsds, at this resolution, should be in the 0.004 to
0.006 A range.  Decreasing the B-factor restraints will help also, but
will over-restrain the side-chains and short loops.  Do not forget to
use TLS, and I would recommend the TLSMD server
(http://skuld.bmsc.washington.edu/~tlsmd/) as an easy and unbiased way
to select your TLS groups.  TLS can model gross B-factor variations with
very few free parameters, and will generally result in a significant
improvement in your model.

Whichever approach you take I wish you good luck, a 3.2 A structure, in
the absence of NCS, can be very difficult to build and refine.

Mark


On Wed, 2007-04-18 at 16:41 +0200, Eva Kirchner wrote:

> Thank you Roberto, I saw that line in the log file, too. So it is as I
> feared: Refmac cannot be stopped from refining B-factors ;-) Maybe
> I'll use CNS...
> 
> Eva
> 
> 
> 2007/4/18, Roberto Steiner < [EMAIL PROTECTED]>:
> 
> 
> On 18 Apr 2007, at 14:39, Eva Kirchner wrote: 
> 
> > 
> 
> 
> 
> 
> 
> > 
> > (But I'm still curious about the B-factor refinement when
> > there is no "REFI BREF ISOT" in the com-file...)  
> 
> 
> 
> Eva, 
> 
> 
> Refmac internal default is REFI BREF ISOT 
> that's why even if you remove the above line from the com file
> (or deselect that option from the interface) it still does
> ISOT BREF refinement. 
> It does tell you that though 
> if you look at the log file there's a bit that says 
> 
> 
> ... 
>  Method of minimisation : Sparse Matrix 
>   Experimental sigmas used for weighting 
>   Number of Bins and width:20   0.0080 
>   Refinement of individual isotropic Bfactors 
> .. 
> what do you have there when you deselect the B fact option
> from the interface? 
> 
> 
> 
> 
> I agree with Herman that at 3.2 A isotropic B values
> refinement can be useful. 
> 
> 
> 
> 
> Roberto 
> 
> 
> > 
> > Eva
> > 
> >  
> > 
> > 2007/4/18, Mischa Machius
> > <[EMAIL PROTECTED]>: 
> > 
> > Like Harry said, it is not justified to do
> > individual B factor refinement at that resolution.
> > Well, you can do it, but you'll end up with funny
> > results, such as what are observing right now.
> > Still, from a pragmatic point of view, individual B
> > factor refinement in cases 

Re: [ccp4bb] Stop Refmac from refining B factors?

[Ethan Merritt]

On Wednesday 18 April 2007 07:40, Phil Jeffrey wrote:
> Harry M. Greenblatt wrote:
> 
> >   You should be refining an overall temperature factor at that 
> > resolution.  It's one of the choices in the list, instead of "isotropic".
> 
> I disagree with this.  At that (3.2 Angstrom) resolution I've often 
> found than a tightly restrained individual B-factor refinement gives a 
> significantly lower R-free than a single overall B-factor.  I also 
> prefer it to grouped B-factors in CNS

My suggestion (as you can probably guess):

Step 1
Set the geometric restraints very tight, but the thermal restraints
not so tight. Refine individual or group B's (I doubt it matters which).

Step 2
Take that model, unrealistic as it is, and feed it to the TLSMD server
for automated analysis of the distribution of B factors in real space.
Tell the server to use the backbone B's only (it's a tick-box on the
'advanced options' page). The output should suggest to you which portions
of your structure are suitable for treatment as coherent groups;
some will be relatively well-ordered, others may be waving around wildly.

Step 3
Go back to refmac and perform a multi-group TLS refinement using the
groups suggested by TLSMD.  Because this is low resolution, you should
refine a pure TLS model.  That is, reset all the individual B's to
some constant value and let the TLS model alone describe their variation.

Success is never guaranteed, but in test cases we have found that for
low resolution structures a multi-group pure TLS model can give better
R values than individual isotropic B refinement, while ensuring that
the distribution of B's adheres to a physically plausible model.

Ethan Merritt







> , because the latter are not  
> geometrically restrained and show a lot of physically unreasonable 
> waywardness (although often, similar R-free as B-individual). 
> Individual B's can also be restrained by non-crystallographic symmetry 
> and as far as I can tell grouped B's are not.
> 
> I think one has to explore all possibilities rather than take one fixed 
> approach to working at modest resolutions, and the optimal solution is 
> likely to be different for different structures.
> 
> Phil Jeffrey
> Princeton, NJ
> 
> 
> 
> >> 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
> > 

> 

-- 
Ethan A MerrittCourier Deliveries: 1959 NE Pacific
Dept of Biochemistry
Health Sciences Building
University of Washington - Seattle WA 98195-7742

Re: [ccp4bb] Stop Refmac from refining B factors?

[Eva Kirchner]

Thank you Roberto, I saw that line in the log file, too. So it is as I
feared: Refmac cannot be stopped from refining B-factors ;-) Maybe I'll use
CNS...

Eva


2007/4/18, Roberto Steiner < [EMAIL PROTECTED]>:



On 18 Apr 2007, at 14:39, Eva Kirchner wrote:





(But I'm still curious about the B-factor refinement when there is no "REFI
BREF ISOT" in the com-file...)


Eva,

Refmac internal default is REFI BREF ISOT
that's why even if you remove the above line from the com file (or
deselect that option from the interface) it still does ISOT BREF refinement.

It does tell you that though
if you look at the log file there's a bit that says

...
 Method of minimisation : Sparse Matrix
  Experimental sigmas used for weighting
  Number of Bins and width:20   0.0080
  Refinement of individual isotropic Bfactors
..
what do you have there when you deselect the B fact option from the
interface?


I agree with Herman that at 3.2 A isotropic B values refinement can be
useful.


Roberto


Eva



2007/4/18, Mischa Machius < [EMAIL PROTECTED]>:
>
> Like Harry said, it is not justified to do individual B factor
> refinement at that resolution. Well, you can do it, but you'll end up with
> funny results, such as what are observing right now. Still, from a pragmatic
> point of view, individual B factor refinement in cases like these can have a
> positive effect on the electron density. However, keep in mind that the
> resulting B factors may physically not be very meaningful. In the end,
> you'll have to switch to grouped B factor refinement, or you risk nasty
> comments from an attentive mentor or reviewer (and rightly so). Hope that
> helps. Best - MM
> On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote:
>
> 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
>
>
>
>
> 

> Mischa Machius, PhD
> Associate Professor
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.; ND10.214A
> Dallas, TX 75390-8816; U.S.A.
> Tel: +1 214 645 6381
> Fax: +1 214 645 6353
>
>
>

 ---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

Re: [ccp4bb] Stop Refmac from refining B factors?

[Phil Jeffrey]

Harry M. Greenblatt wrote:

  You should be refining an overall temperature factor at that 
resolution.  It's one of the choices in the list, instead of "isotropic".


I disagree with this.  At that (3.2 Angstrom) resolution I've often 
found than a tightly restrained individual B-factor refinement gives a 
significantly lower R-free than a single overall B-factor.  I also 
prefer it to grouped B-factors in CNS, because the latter are not 
geometrically restrained and show a lot of physically unreasonable 
waywardness (although often, similar R-free as B-individual). 
Individual B's can also be restrained by non-crystallographic symmetry 
and as far as I can tell grouped B's are not.


I think one has to explore all possibilities rather than take one fixed 
approach to working at modest resolutions, and the optimal solution is 
likely to be different for different structures.


Phil Jeffrey
Princeton, NJ




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


-

Harry M. Greenblatt

Staff Scientist

Dept of Structural Biology   [EMAIL PROTECTED] 



Weizmann Institute of SciencePhone:  972-8-934-3625

Rehovot, 76100   Facsimile:   972-8-934-4159

Israel 

Re: [ccp4bb] Stop Refmac from refining B factors?

[Roberto Steiner]

On 18 Apr 2007, at 14:39, Eva Kirchner wrote:







(But I'm still curious about the B-factor refinement when there is  
no "REFI BREF ISOT" in the com-file...)


Eva,

Refmac internal default is REFI BREF ISOT
that's why even if you remove the above line from the com file (or  
deselect that option from the interface) it still does ISOT BREF  
refinement.

It does tell you that though
if you look at the log file there's a bit that says

...
Method of minimisation : Sparse Matrix
  Experimental sigmas used for weighting
  Number of Bins and width:20   0.0080
  Refinement of individual isotropic Bfactors
..
what do you have there when you deselect the B fact option from the  
interface?



I agree with Herman that at 3.2 A isotropic B values refinement can  
be useful.



Roberto



Eva



2007/4/18, Mischa Machius <[EMAIL PROTECTED]>:
Like Harry said, it is not justified to do individual B factor  
refinement at that resolution. Well, you can do it, but you'll end  
up with funny results, such as what are observing right now. Still,  
from a pragmatic point of view, individual B factor refinement in  
cases like these can have a positive effect on the electron  
density. However, keep in mind that the resulting B factors may  
physically not be very meaningful. In the end, you'll have to  
switch to grouped B factor refinement, or you risk nasty comments  
from an attentive mentor or reviewer (and rightly so). Hope that  
helps. Best - MM


On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote:


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



-- 
--

Mischa Machius, PhD
Associate Professor
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353





---
Dr. Roberto Steiner
Randall Division of Cell and Molecular Biophysics
New Hunt's House
King's College London
Guy's Campus
London, SE1 1UL
Phone +44 (0)20-7848-8216
Fax   +44 (0)20-7848-6435
e-mail [EMAIL PROTECTED]

[ccp4bb] AW: [ccp4bb] Stop Refmac from refining B factors?

[Herman . Schreuder]

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 
), 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 SciencePhone:  972-8-934-3625

Rehovot, 76100   Facsimile:   972-8-934-4159

Israel 

Re: [ccp4bb] Stop Refmac from refining B factors?

[Eva Kirchner]

Thank you both, Mischa and Harry, I'll try overall B refinement. But I'm
starting to think that my Refmac is somewhat crappy, I tried this before,
and then all the B-factors were exactly the same. Now, I tried it again and
they are not... Something's still wrong here.

(But I'm still curious about the B-factor refinement when there is no "REFI
BREF ISOT" in the com-file...)

Eva



2007/4/18, Mischa Machius <[EMAIL PROTECTED]>:


Like Harry said, it is not justified to do individual B factor refinement
at that resolution. Well, you can do it, but you'll end up with funny
results, such as what are observing right now. Still, from a pragmatic point
of view, individual B factor refinement in cases like these can have a
positive effect on the electron density. However, keep in mind that the
resulting B factors may physically not be very meaningful. In the end,
you'll have to switch to grouped B factor refinement, or you risk nasty
comments from an attentive mentor or reviewer (and rightly so). Hope that
helps. Best - MM
On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote:

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





Mischa Machius, PhD
Associate Professor
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.; ND10.214A
Dallas, TX 75390-8816; U.S.A.
Tel: +1 214 645 6381
Fax: +1 214 645 6353

Re: [ccp4bb] Stop Refmac from refining B factors?

[Harry M. Greenblatt]

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  
), 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 SciencePhone:  972-8-934-3625
Rehovot, 76100   Facsimile:   972-8-934-4159
Israel

[ccp4bb] Stop Refmac from refining B factors?

[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