On 03/01/2021 01:50, Emilia C. Arturo (Emily) wrote:
Hello all,
Hello Emily,
I suspect that there were no replies to these questions because those
no-one has a sufficiently rounded experience. I know something about
Coot and can make guesses about OneDep. More details are required.
For relatively low resolution maps, where sugar moieties cannot be
placed unambiguously, in theory (say Agirre(2017) and Emsley and
Crispin (2018)) pyranose placement within the map should be quite
highly restrained by ideal geometry for the specific sugar expected
from prior knowledge. In practice, however, it’s not entirely clear
how to maintain these restraints on pyranose geometry and conformation
during manual building in Coot, or how to apply any special restraints
in Phenix. The Glyco module of Coot does indeed place fine looking
Asn-NAGs after a few iterations of manual placing and refining to
account for the poor resolution of the acetyl group, just as discussed
by Emsley and Crispin (2018). However, the validation report that is
obtained from the OneDep system at the rcsb web server for the
resulting structure shows many outliers in bonds and angles within the
NAG or between the NAG and protein Asn residue. In Coot I have
experimented with various restraints(in the R/RC menu), including
decreasing the Refinement weight/weight matrix to zero, or some small
non-zero number, thinking this places less weight on the map signal
and more on the ideal values within Coot’s libraries. I have tried a
variety of refinement restraint combinations in Coot to address
individual bonds and angles outliers within the sugar, but the rcsb
validation report still finds departures from ideal geometry.
Meanwhile, in Phenix I don’t do anything specific for the refinement
of glycosylation sugars (should I?) other than apply the appropriate
restraints for any low resolution EM map; are there specific
restraints files I ought to be using within Phenix real space refine
jobs for N-linked sugars?
Give us examples - what are the top 3 outliers in bond lengths or
angles? Does the distance from OneDep correspond to the distance in the
model? Why does OneDep think that they out outliers? (i.e. what is the
target distance and sigma? (n_sigma_crit is 3, I presume?)) When Coot
starts it tells you which restraints library it is reading. What does it
say? The NAG-ASN link in mon_lib_list.cif is what Coot uses. If you
don't like it you can use AceDrg to generate a new one then read it into
Coot as you would a monomer restraints dictionary file.
It turns out that regarding higher resolution maps I am also unsure of
exactly how to apply restraints within Coot. For example, using PDB ID
5VAA and its map with a reported resolution of 1.55A, I centered on
Asn297 and its associated glycosylation sugars. I experimented with
refinement strategies (i.e. the settings of R/RC), thinking that the
best approach would be little restraint on ideal geometry and higher
weight on the map signal. But no matter what combination of restraints
I used, I was unable to keep the model locally from distorting; i.e.
the deposited model versus map looks great, but within Coot I find a
hundred ways to destroy it. I must be doing something wrong.
Coot has not been tuned to build N-linked glycans in cryo-EM
reconstructions. Not yet, that is.
Maybe the restraints are fighting the density fit? To test:
R/RC -> Estimate. Multiply that number by 2.5 (say). OK.
Calculate -> Modules -> Carbohydrate
Calculate -> Scripting -> Scheme
(set! *cho-bond-sigma-scale* 33)<Enter>
Now try to build your N-linked glycan. It will behave differently. Maybe
it will no longer listen to the distance restraints.
Maybe there is a happy medium.
Paul
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