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Re: [ccp4bb]: Enforcing a sigma(f) cutoff, reflex reaction to theidea of?

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The words "sigma(F) cutoff" elicit a knee-jerk reaction in some
crystallographers that may make it hard for them to finish reading
a post.  If I carefully scrutinize the message below, reading
between the lines and all that, I get the distinct impression
that this fellow is not solving a new structure at all (his crystals
are still growing, I suspect) but honing his skills in crystallo-
graphic computing by re-solving a structure that was deposited
together with structure factors in the PDB.  He does everything
by the book and he is surprised to find that the R-free
comes out 2 points higher than the value mentioned in the
PDB file. This may indicate he is making a blunder, so he
wants to resolve the discrepancy before starting in on his
own protein.

He already has one reply suggesting the discrepancy is probably
not due to sigma cutoff, and if he accepts that he is no longer
interested in forcing refmac to use a sigma(F) cutoff of 0.000.
Now he needs other suggestions as to what causes the discrepancy.

Mark, Is your R-factor as well as R-free higher than published?
If you use the exact deposited structure including waters,
and do no or minimal (single rigid body) refinement, do you still
get a high R-free? This would indicate the difference lies not in
the structures but in something like bulk solvent or anisotropic
B-factor corrections or in the way R-free is calculated.
Presumably you chose a new Free-R set, which will include
mainly working reflections of the original authors, so your
R-free should be artificially low (the opposite of what you see)
on their refined structure.

Or do you see the discrepancy only when you re-solve the
structure by molecular replacement using a related protein,
in which case your structure may really not be as good as the
published one? Did the original authors have the advantage of
experimental phases, multiple crystal forms, or something?
The PDB deposition only involves the dataset against which the
structure was refined, so that's all you have to work with,
but in reality a lot of other data may have been used in
building and refining the model.


Mark DePristo wrote:
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> Hello,
> I have a general question about refinement with refmac5.  I've been 
> re-solving a protein structure against its structure factors (obtained 
> from PDB).  One consistent difference between my new structure and the 
> PDB structure is that the PDB structure uses a sigma(f) cutoff of 0.0, 
> as seen in the REMARK in the pdb file:
> REMARK   3   DATA CUTOFF            (SIGMA(F)) : 0.000
> My structure's PDB file, as generated by refmac5, has the line
> REMARK   3   DATA CUTOFF            (SIGMA(F)) : NONE
> For the life in me I cannot figure out how to enforce a sigma(f) cutoff 
> in refmac5.  There are some suggestions (in the archives of ccp4bb) that 
> such a cutoff can introduce artifacts, as the sigma(f) cutoff 
> disproportionately affects the weaker reflections.  Regardless, I am 
> suspicious that this cutoff accounts for the slightly higher R and 
> R-Free values (~ 2%) of my new structure.  Is this a reasonable 
> interpretation?  If so, how can I enforce a sigma(f) data cutoff?
> Thanks,
> Mark
> Mark DePristo
> Ph.D. Candidate
> Dept. of Biochemistry
> Cambridge University
> mdepristo@cryst.bioc.cam.ac.uk
> http://www-cryst.bioc.cam.ac.uk/~mdepristo/

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