[phenixbb] CaBLAM fixing and interpretation in comprehensive validation for cryo-EM
christopher.sci.williams at gmail.com
Fri Jun 28 12:50:13 PDT 2019
[I forgot to copy my reply to the bulletin board, so here it is, reproduced
for the record.]
The official publication for CaBLAM is the 2017 Molprobity paper in Protein
Science, here: https://doi.org/10.1002/pro.3330 Further technical
documentation on CaBLAM can be found in Phenix's Computational
Crystallography Newsletter, here:
recommend the newsletter article as a fast read.
To identify outliers, CaBLAM looks at a structure's CA trace, which is
generally well-modeled. For each residue, it compares the local peptide
plane orientations of the model to the observed distribution of peptide
plane orientations for high quality residues with matching CA trace
geometry. The CaBLAM score is a percentile score that rates how well the
model matches with the expected distribution. The lower the score, the
rarer the observed conformation is in our database of quality structures.
A conformation falling in the bottom 5% of observed behavior is potentially
suspicious ("Disfavored") and a conformation falling in the bottom 1% is
considered an outlier.
This percentile-based scoring is fundamentally the same scoring used in
MolProbity's description of Ramachandran and rotamer outliers, though of
course CaBLAM puts its cutoffs in different places.
As a matter of interpretation, loop/coil regions tend to be highly varied.
CaBLAM "Disfavored" conformations in loops can largely be ignored.
However, disfavored conformations in regions expected to by highly regular
(repeating secondary structure) should be taken seriously. CaBLAM outliers
should be inspected wherever they occur.
The CA geometry score looks at just the CA trace, and takes the CA virtual
angle into account (defined by CAi-1, CAi, CAi+1). Outliers in this space
reflect some sort of severe problem with CA geometry, often involving an
over-extended or over-compressed CA virtual angle.
The secondary structure scores are based on how well a residue's local CA
trace matches the expected CA trace of each major secondary structure type,
alpha, 3-10, and beta. You can see the contours used for this assessment
in Figure 3 of the newsletter article. Each residue receives individual
secondary structure scores. Then regions of residues that all pass a
scoring threshold are assembled into probable secondary structure
elements. This is where the "try beta sheet" recommendations come from.
That recommendation indicates that the residue in question *and its
neighboring residues* all have CA traces that look like beta sheet.
I wish I had a simple recommendation for you, but fixing CaBLAM outliers
systematically has proven to be a challenge. Take a look at your structure
and see if you believe that the outlier residues really are intended to be
part of beta sheets. If so, beta sheets have distinctive hydrogen bonding
patterns that tend to be disrupted by the kind of problems that CaBLAM
identifies. Ideally, you will be able to use Coot's tools to restore the
proper hydrogen bonding. Then, applying hydrogen bonding restraints during
refinement may help keep your work in place.
If you have large regions of outliers, it may instead be more practical to
strip out the existing model and replace it with idealized beta sheet
structure, then rerefine. Again, hydrogen bonding restraints may be
As a general rule, CaBLAM outliers usually indicate a problem with the
orientations for one or more peptide planes. Look for a way to reorient
the peptide either to remove clashes or establish hydrogen bonds. Make
sure you build good regular secondary structure, don't sweat about the
loops too much, and trust your judgement and experience to identify the
real and justified outliers.
We of the Richardson Lab generally dislike torsion-based Ramachandran
restraints/secondary structure restraints. It's very easy to accidentally
generate a model that looks better than it really is using these methods.
However, we recognize that these are powerful tools for a difficult problem
and you may get good out of careful use.
Hope that helps, and good luck
---Richardson Lab, Duke University
On Wed, Jun 26, 2019 at 2:21 PM Ahmad Khalifa <underoath006 at gmail.com>
> I would appreciate some insights on how to interpret the validation
> results under the CaBLAM tab and how to use it to fix my structure in Coot?
> What are the CaBLAM score, the Ca geometry score, the helix score and the
> 3-10 score?
> How to use the information under the secondary structure tab? In my
> protein for example, I see a lot of "try beta sheets", should I try to
> refine that segment using beta sheets restraints?
> Best regards.
> phenixbb mailing list
> phenixbb at phenix-online.org
> Unsubscribe: phenixbb-leave at phenix-online.org
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