On 04/11/2023 14:04, Sam Tang wrote:
Dear community,
I am solving the structure of a complex between proteins A and B,
where A is a protein with known homologs and B is a novel protein
isolated from plant. The diffraction data was at 1.9 Ang collected
in-house, indexed to P321. Using A as the search model, we have got a
reasonable solution where, after one round of refinement, the A chain
fits the map pretty well. What's left was to extend the termini and
fit a few rotamers.
For protein B (B chain) I have tried the web version of ARP/wARP but
the outcome was not really good. The model was not successfully built
as indicated by low model completeness and score. The tricky thing may
be that we do not have the complete sequence information of this
protein B in-hand. (The other way round, we more or less wish to rely
on the high resolution data to confirm its sequence.) What approach
would you then recommend to build the B chain in this scenario?
There are two steps:
(i) get poly-ALA trace fragments for B
(ii) convert poly-ALA fragments to true sequence
If you want to use Coot:
You can do the former by placing a helix and extending it by "Add
Terminal Residue" (hot-key "Y") or (as you saw) by running APR/wARP
ModelCraft or res_tracer (leave the pir filename as blank of course).
You can do the latter by using
(a) the tool in Cryo-EM module "Auto-assign sequence based on map"
and then BLASTing that sequence (or set of sequences, one presumes)
once you have the correct sequence then use "Align and Mutate" or
(b) export the poly-ALA model and use coot-identify-protein using
each of the fragments in your poly-ALA and the genome of you organism
(in FASTA format). That will tell you the sequence of the
fragment so that you can then "Mutate Sequence."
If you can't see at least tubes for the missing chain, then this
approach is hopeless.
Paul.
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