Re: Re: [ccp4bb]: Symmetry breakage, twinning, high b's ...

["Jorge Iulek" <iulek@interponta.com.br>]

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Dear colleagues,

    Many thanks for the several replies.
    Although all of them were sent to the bb itself, as solicited I am
making a general summary, also adding some information which were asked for.
Please, write me if the new informations rise some more suggestions.
    Stephen Soisson (SS), Eleanor Dodson (ED), Mischa Machius (MM),
Raaijmakers (R) and Anastassis Perraskis (AP) answered.
    The main points: biological dimer, data processed in P6122, 1 monomer
au, anisotropy of intensity distribution, refinement stuck at
"Overall R factor = 0.2258 Free R factor = 0.2873",
then reprocessed in P61, 2 monomer au, no more anisotropy in intensity
distribution,
initial refinement at
"Overall R factor= 0.2067 Free R factor = 0.2746"
    In both P6122 and P61, regions of bad fitting between model and
density, also B's ~41 AČ. But in P61, significative diferences in the AMP
ligand density between the dimmers, but only there.

1) MM and AP point that they would be happy if some of their structures
got stuck at those R's for P6122.
2) SS, MM and ED ask for the B's from the Wilson Plot (my faul not to
inform).
In P6122, it is 40.722 and in P61 it is 40.360, so very close to the average
I am getting in the refinement. Would them be this such strong indicative
that
a similar value should be gotten during my refinement ?

Crystal was cryocooled (100 K), but has 53 % solvent. Maybe the B's reflect
an internal disorder
(hum, some other similar crystal did not diffract at all...) ?

3) SS suggests to set all B's of the model to the one from data, then
simulated annealing (also
to take out bias from model).
4) MM says <B> ~40 A is not so unusual.
5) R suggests TLS refinement (each hellix as a domian). He had a similar (B
and
R's) case where this improved (attacking flexibility of the protein).
6) AP points to an article with high R_free. Funny the anisotropy, he asks
for
other indicatives of twinning.
    As far as I can understand, no way (symmetry operator) for twinning in
P6122. I tested for P61 and, as expected, almost 50 % twinned (data accepts
to be processed in P6122 with low R_merge ...).
7) ED made several suggestions and notes:
    "Due to ML, have to look at 1 sigma..."
    Yes, I am using ML.
     "... spikes of relatively hihg B..."
    There are B's at 50, 60 and even at 80 AČ. But I should check better,
probably  the higher ones at flexible regions...
     "...looked at truncate 2nd moments for acentric reflections..."
    Yes, I did. No, they are quite close to 2 and even a bit higher. So,
this does not indicate twinning
    ".. leave the suspect atoms out altogether - then redo  the maps.. "
    I have to make this to confirm symmetry breakage at looking to the
maps.
    Also, ED answered to the main question:
> 3) In the case of P61, at using two monomers, I double the number of
> parameters. Of course this
> should allow an artificial lowering of the R factor, but I also doubled
the
> number of reflections (hum...., although they are quite similar pairwise).
> Who weights more here ? Doubled parameters, doubled data ? Should the R
> artificially lower, raise or keep the same ? But, in the case where the
data
> are similar pairwise, should this not be decisive for artificially
lowering
> the R ?

 "Free R goes down too - meant to mean things are improving.."

    So I conclude that the lower R-free does indicate I should refine my
structure in P61 (and this way publish it), Okay ?

    Finally, ED suggests me to refine the initial MR model with my R_free
flagged set (but their F's, of course), using my protocol and compare the
results.

    Many thanks to you all. I hope I could summarize without distorting
words.
    Whether anyone else has other advices, they would be welcome (e.g.,
 what would be the products of AMP degration ?). Send me
privately if you wish .


Jorge


************************************************************************
Prof. Jorge Iulek, Ph. D.
Protein Purification and 3D Structure Determination Group
Department of Chemistry
State University of Ponta Grossa - PR
Brazil

Office phone: ++ 55 (42) 220-3062
Home phone  : ++ 55 (42) 224-6182
Fax  : ++ 55 (42) 220-3342


e-mail: iulek@interponta.com.br
              iulek@uepg.br
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