| Dear Colleagues, I think this paper from 1979 is still very interesting:- John Emeritus Professor John R Helliwell DSc
Hello:yes, a great discussion! I second Eleanor's statement that B-factors of high resolution structures do carry a message about atom flexibility. I attach a screenshot of a figure from our paper (Schneider et al.: Local dynamics of proteins and DNA evaluated from crystallographic B factors, Acta Cryst. (2014). D70, 2413–2419) that shows clear resolution dependence of B factors at protein/protein interface for amino acids and waters. Our high resolution group of structures could not be below 1 Å as Eleanor suggests but even modest limit to 1.9 Å and then structures at 1.9-2.5 and 2.5-3.0 show the effect clearly. We looked at several other groups of atoms (backbone/side chains at the protein core, at the protein surface, DNA phosphates/bases, waters at the interfaces or bound on the protein surface) and saw the same dependence.Best,Bohdan, bs.structbio.orgOn 2024-08-02 13:26, Eleanor Dodson wrote:All interesting points.. (And good to see a reference to /" P.A. Machin, J.W. Campbell, M. Elder (Eds)
Refinement of Protein Structures, SERC Daresbury Laboratory, Warrington, UK (1980)"/
- for those who remember, a super exciting discussion over what was feasible for refinement, and how to do it! )
My take - if a crystal diffracts to 1A we can be fairly sure of the accurate position of most of the coordinates, see other conformations for some regions, and give realistic B values to most atoms.
If the crystal only diffracts to 3A then the lattice is not perfect, and there must be multiple conformations for lots of the molecule.
There is not going to be sufficient experimental data to model this properly so every parameter assuming a single conformer - coordinate, B value, occupancy - is an approximation. Restraints help to some extent but they impose prior knowledge and do not glean information from the experimental data.
The "trash can" should indicate the degree of uncertainty and interpreting that is a bit problematic. B values twice the overall B ?? Hmm- do NOT base too much faith in that part of the model.. As crystallographers I think maybe we need to flag this better for trusting users of the information. Omitting that region? I am not sure .. How do others model those floppy lysines? I usually make a sort of informed guess but indeed giving a single conformation is not the truth, the whole truth, and nothing but the truth..
On Fri, 2 Aug 2024 at 01:14, James Holton <[email protected]> wrote:
__
I submit that modern B factor restraints make them much less trashy
than they were in the early days. As Pavel points out the exact
strategies differ from program to program, but I don't think anybody
does unrestrained B factor refinement. Not by default.
Besides, all we are really doing is fitting Gaussian-shaped peaks to
the "curve" of the data. These peaks have a width and a height. For
example, a carbon atom with B=20 has a peak density of 1.6 e-/A^3
and a full-width-at-half-max (FWHM) of 1.4 A. That is it! That is
the model density being fit. If you increase to B=80 the peak drops
to 0.3 e-/A^3 and the FWHM increases to 2.6 A. At the largest B you
can stuff into a PDB file (999.99), the peak height is 0.008 e-/A^3
and the "peak" is 8.45A wide. Your disordered loop, however, is
probably not sampling from a symmetric Gaussian distribution like
that. This is the real problem with large B factors. They can fit
better than sharper B atoms, but that doesn't mean they fit well.
Occupancy is easy because all it does is scale the height without
affecting the width. So, an 0.5 occupancy atom model is half the
height of a full-occupancy one. The width is unchanged. B factors
impact both width and height because they must preserve the number
of electrons in the peak. This is perhaps why they are often
confusing and mysterious. We should also never forget that bulk
solvent gets excluded with exactly the same radii rules from every
modeled atom, regardless of B factor and occupancy. So, the "change
in density" from adding or deleting an atom is a little more
complicated than adding or subtracting a Gaussian peak.
Nevertheless, if you want to fit peak height and width independently
(like we do in pretty much every other kind of curve fitting), then
you should refine occupancy and B factors at the same time.
Over-fitting you say? Hardly. Polynomials are easy to over-fit, but
not Gaussians. Observations/parameters is a useful guide for
polynomial fits, but in general the hallmark of over-fitting is that
the prediction passes exactly through all the observed points (and
not the cross-validation or "Rfree" points). I have never seen a
macromolecular refinement end up with Rwork = 0. Have you?
At the end of the day, what we do with our models is look at their
parameters and try to extract the physically meaningful reality they
are trying to capture. Restraints are very helpful in preventing
many types of unrealistic situations, but ultimately it is up to you
to decide if the fitted model makes sense.
-James Holton
MAD Scientist
On 7/30/2024 11:30 AM, Ian Tickle wrote:
Obviously no refined parameters can ever be completely error-free,
it's just that for the co-ordinates we have very accurate
geometric restraints so that the relative uncertainty in the
refined co-ordinates is small (but try refining co-ordinates
without restraints!). For the B factors we don't have accurate
estimates (if any) for their restraints so their relative
uncertainty after refinement is much greater.
-- Ian
On Tue, Jul 30, 2024 at 6:57 PM Oganesyan, Vaheh <[email protected]> wrote:
Yes, it is and I like the definition of shared “trash bin”. It
will have more physical meaning if we can separate those
contributions into separate bins.
Vaheh
*From:* Pavel Afonine <[email protected]
<mailto:[email protected]>>
*Sent:* Tuesday, July 30, 2024 1:51 PM
*To:* Oganesyan, Vaheh <[email protected]
<mailto:[email protected]>>
*Cc:* [email protected] <mailto:[email protected]>
*Subject:* Re: [ccp4bb] How high a B factor is too high to
assume a loop is in place, in the AlphaFold era?
Vaheh,
I think coordinates are no different from B factors,
occupancies, f', or f'' in this respect. Coordinates can play
their "trash bin" role by adjusting to the noise at the
expense of violated geometry (bonds, angles, planes, torsions,
etc.). As I mentioned in my previous email, their trash bin
capacity is much smaller (but definitely not zero!) because
the number and strength (confidence) of geometry restraints
are much greater than those of ADP restraints.
I agree that all refined parameters share this trash bin
capacity, but to varying degrees. Isn't this essentially what
we call the error on the refined parameter? All refined
parameters have their error bars, which we have referred to as
the "trash bin" in this thread.
Pavel
On Tue, Jul 30, 2024 at 10:09 AM Oganesyan, Vaheh
<[email protected]> wrote:
Your point is taken, Pavel. However, despite resolution,
you define coordinate of the atom as a geometric point
with no width. Although coordinates are “refineable”, they
have no capacity for “trash”. Their “trash” still goes
into B-factor “trash bin”. At least this is how I see it.
Thank you.
*Vaheh Oganesyan, Ph.D.*
*R&D **| Biologics Engineering*
One Medimmune Way, Gaithersburg, MD 20878
T: 301-398-5851
[email protected]
*From:* Pavel Afonine <[email protected]>>
*Sent:* Tuesday, July 30, 2024 11:45 AM
*To:* Oganesyan, Vaheh <[email protected]>
*Cc:* [email protected] <mailto:[email protected]>
*Subject:* Re: [ccp4bb] How high a B factor is too high to
assume a loop is in place, in the AlphaFold era?
From this perspective, all refinable atomic model
parameters can be viewed as trash bins, with the size of
these bins being proportional to the amount of prior
information (restraints) imposed on these parameters. For
example, coordinates have the most restraints and thus are
the smallest trash bins, while B factors have the least
restraints and thus are one of the largest bins.
Pavel
On Tue, Jul 30, 2024 at 8:25 AM Oganesyan, Vaheh
<[email protected]> wrote:
Early in my Crystallography life I was postdoc with
Robert Huber in Munich. We had those gatherings once a
week when in very informal way we can ask and answer
questions. I remember my question about B factors: how
is it possible to have high resolution structure and
average B-factor of 100A^2 . I think it was Robert or
Albrecht Messerschmidt who told that B-factor is a
“trash can” that describes not only loosely positioned
atoms but also all other problems that either you
created during processing, harvesting or crystal had
from the beginning.
*Vaheh Oganesyan, Ph.D.*
*R&D **| Biologics Engineering*
One Medimmune Way, Gaithersburg, MD 20878
T: 301-398-5851
[email protected]
*From:* CCP4 bulletin board <[email protected]> *On Behalf Of *James
Holton
*Sent:* Tuesday, July 30, 2024 10:35 AM
*To:* [email protected] <mailto:[email protected]>
*Subject:* Re: [ccp4bb] How high a B factor is too
high to assume a loop is in place, in the AlphaFold era?
How high B factors can go depends on the refinement
program you are using.
In fact, my impression is that the division between
the "let the B factors blow up" and "delete the
unseen" camps is correlated to their preferred
refinement program. You see, phenix.refine is
relatively aggressive with B factor refinement, and
will allow "missing" atoms to attain very high B
factors. Refmac, on the other hand, has restraints
that try to make B factor distributions look like
those found in the PDB, and so tends to keep nearby B
factors similar. As a result, you may get "red
density" for disordered regions from refmac, inviting
you to delete the offending atoms, but not from
phenix, which will raise the B factor until the
density fits.
Then there are programs like VagaBond that don't
formally have B factors, but rather let an ensemble of
chains spread out in the loopy regions you are
concerned about. This might be the way to go?
You can also do ensemble refinement in the latest
Amber. That is, you run an MD simulation of a unit
cell (or more) and gradually increase structure factor
restraints. This would probably result in the "fan" of
loops you have in mind?
-James Holton
MAD Scientist
On 7/28/2024 8:13 AM, Javier Gonzalez wrote:
Dear CCP4bb,
I'm refining the ~3A crystal structure of a big
protein, largely composed of alpha helices
connected by poorly-resolved loops.
In the old pre-AlphaFold (AF) days I used to
simply remove those loops/regions with too high B
factors, because there was little to none density
at 1 sigma in a 2Fo-Fc map.
However, considering that the quality of a
readily-computable AF model is comparable to a 3A
experimental structure, and that the UniProt
database is flooded with noodle-like AF models, I
was considering depositing a combined model in the
PDB.
Once R/Rfree reach a minimum for the model
truncated in poorly resolved loops, I would
calculate an augmented model with AF calculated
missing regions (provided they have an acceptable
pLDDT value), assign them zero occupancy, and run
only one cycle of refinement to calculate the
formal refinement statistics.
Would that be acceptable? Has anyone tried a
similar approach?
I'd rather do that instead of depositing a
counterintuitive model with truncated regions that
few people would find useful!!
Thank you for your comments,
Javier
-- Dr. Javier M. González
Instituto de Bionanotecnología del NOA
(INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
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