Dale Tronrud wrote:
> While what you say here is quite true and is useful for us to
> remember, your list is quite short. I can add another
>
> 3) The systematic error introduced by assuming full occupancy for all
> sites.
You are right that structural heterogeneity is an additional factor.
SeMet expression is one of the examples where the SeMet residue is
often not fully incorporated, and therefore its side chains have mixed
with Met composition.
Obviously, solvent molecules may have partial occupancies.
Also, in heavily exposed crystals chemical reactions result in loss of
the functional groups (e.g. by decarboxylation).
However, in most cases even if side chains have multiple conformations
their total occupancy is 1.0.
>
> There are, of course, many other factors that we don't account for
> that our refinement programs tend to dump into the B factors.
>
> The definition of that number in the PDB file, as listed in the mmCIF
> dictionary, only includes your first factor 
>
> http://mmcif.rcsb.org/dictionaries/mmcif_std.dic/Items/_atom_site.B_iso_or_equiv.html
>
>
> and these numbers are routinely interpreted as though that definition is
> the law. Certainly the whole basis of TLS refinement is that the B factors
> are really Atomic Displacement Parameters. In addition the stereochemical
> restraints on B factors are derived from the assumption that these
> parameters
> are ADPs. Convoluting all these other factors with the ADPs causes serious
> problems for those who analyze B factors as measures of motion.
>
> The fact that current refinement programs mix all these factors with the
> ADP for an atom to produce a vaguely defined "B factor" should be
> considered
> a flaw to be corrected and not an opportunity to pile even more factors
> into
> this field in the PDB file.
>
Bfactors describe overall uncertainty of the current model. Refinement
programs, which do not introduce or remove parts of the model (e.g. are
not able to add additional conformations) intrinsically pile up all
uncertainties into Bfactors. Solutions, which you would like to see
implemented, require a modelbuilding like approach. The test of the
success of such approach would be a substantial decrease of Rfree
values. If anybody can show it, it would be great.
Zbyszek
> Dale Tronrud
>
> On 3/31/2011 9:06 AM, Zbyszek Otwinowski wrote:
>> The Bfactor in crystallography represents the convolution (sum) of two
>> types of uncertainties about the atom (electron cloud) position:
>>
>> 1) dispersion of atom positions in crystal lattice
>> 2) uncertainty of the experimenter's knowledge about the atom position.
>>
>> In general, uncertainty needs not to be described by Gaussian function.
>> However, communicating uncertainty using the second moment of its
>> distribution is a widely accepted practice, with frequently implied
>> meaning that it corresponds to a Gaussian probability function. Bfactor
>> is simply a scaled (by 8 times pi squared) second moment of uncertainty
>> distribution.
>>
>> In the previous, long thread, confusion was generated by the additional
>> assumption that Bfactor also corresponds to a Gaussian probability
>> distribution and not just to a second moment of any probability
>> distribution. Crystallographic literature often implies the Gaussian
>> shape, so there is some justification for such an interpretation, where
>> the more complex probability distribution is represented by the sum of
>> displaced Gaussians, where the area under each Gaussian component
>> corresponds to the occupancy of an alternative conformation.
>>
>> For data with a typical resolution for macromolecular crystallography,
>> such multiGaussian description of the atom position's uncertainty is not
>> practical, as it would lead to instability in the refinement and/or
>> overfitting. Due to this, a simplified description of the atom's position
>> uncertainty by just the second moment of probability distribution is the
>> right approach. For this reason, the PDB format is highly suitable for
>> the
>> description of positional uncertainties, the only difference with other
>> fields being the unusual form of squaring and then scaling up the
>> standard
>> uncertainty. As this calculation can be easily inverted, there is no loss
>> of information. However, in teaching one should probably stress more this
>> unusual form of presenting the standard deviation.
>>
>> A separate issue is the use of restraints on Bfactor values, a subject
>> that probably needs a longer discussion.
>>
>> With respect to the previous thread, representing poorlyordered (so
>> called 'disordered') side chains by the most likely conformer with
>> appropriately high Bfactors is fully justifiable, and currently is
>> probably the best solution to a difficult problem.
>>
>> Zbyszek Otwinowski
>>
>>
>>
>>>>>  they all know what B is and how to look for regions of high B
>>>>> (with, say, pymol) and they know not to make firm conclusions about
>>>>> Hbonds
>>>>> to flaming red side chains.
>>>>
>>>> But this "knowledge" may be quite wrong. If the flaming red really
>>>> indicates
>>>> large vibrational motion then yes, one whould not bet on stable
>>>> Hbonds.
>>>> But if the flaming red indicates that a wellordered sidechain was
>>>> incorrectly
>>>> modeled at full occupancy when in fact it is only present at
>>>> halfoccupancy
>>>> then no, the Hbond could be strong but only present in that
>>>> halfoccupancy
>>>> conformation. One presumes that the other halfoccupancy location
>>>> (perhaps
>>>> missing from the model) would have its own Hbonding network.
>>>>
>>>
>>> I beg to differ. If a side chain has 2 or more positions, one should
>>> be a
>>> bit careful about making firm conclusions based on only one of those,
>>> even
>>> if it isn't clear exactly why one should use caution. Also, isn't the
>>> isotropic B we fit at "medium" resolution more of a "spherical cow"
>>> approximation to physical reality anyway?
>>>
>>> Phoebe
>>>
>>>
>>>
>>
>>
>> Zbyszek Otwinowski
>> UT Southwestern Medical Center at Dallas
>> 5323 Harry Hines Blvd.
>> Dallas, TX 753908816
>> Tel. 2146456385
>> Fax. 2146456353
>

Zbyszek Otwinowski
UT Southwestern Medical Center
5323 Harry Hines Blvd., Dallas, TX 753908816
(214) 645 6385 (phone) (214) 645 6353 (fax)
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