Hi Matthew, As Folmer points out, if the 16 copies of your molecule are all the same, it is most convenient to work on a single copy and then apply symmetry to generate the other 15 copies. In Phenix you can get the symmetry operations relating your 16 chains with the "Find NCS operators" (phenix.find_ncs) tool. This will also tell you how similar the chains are. You can select one chain with the "PDB tools" (phenix.pdbtools) method and edit that one chain. Then you can use the "Apply NCS operators" (phenix.apply_ncs) tool to generate all 16 chains from the one that you have worked on. Now all 16 chains will be identical. If your 16 copies are different, you might still want to do something similar. You could (1) get the NCS operators as described above, (2) build your 30 residues for one chain, (3) apply the NCS operators to the 30 residues alone. These might not join properly for all of the 16 copies and you might have to do some refinement or manual adjustments. All the best, Tom T On Tue, Aug 17, 2021 at 1:24 AM Folmer Fredslund wrote:

Dear Matthew,

You say you have 16 identical chains? Is the electron density also identical between them? How close are the NCS related copies?

I would choose to use Coots built-in NCS tools to copy the model from one chain to the others. Takes a few seconds tops, and then normal refinement should take care of the rest.

See e.g. here

https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/docs/coot.html#Appl...

Unless of course all these N-terminal residues are rather different, then it's a different story, but not quite sure about it from your description.

Hope this helps

Folmer

tir. 17. aug. 2021 08.16 skrev Whitley, Matthew J :

...

Hello all,

I have a very large protein with 16 copies in the asymmetric unit (nearly 15,000 total residues). Upon initial model building, ~30 residues at the N-terminus of each identical chain were not built. After building the reliable parts of the model and filling in other gaps and then refining that partial (but nearly complete) model, the N-terminal density has now improved to the degree that I can manually build a number of the missing residues in Coot. However, that means manually building the same series of residues over and over for each of the 16 identical chains. Is there a way to get AutoBuild to add these N-terminal residues for me to spare me the tedium of repetitive manual building?

What I’ve gleaned from the documentation so far:

From the phenix.fit_loops documentation, it seems like that tool only works for filling in chain breaks, i.e. there is already modeled protein at each end of the missing region. That’s not the case for me because I’m trying to work backward toward the N-terminus.

If I am reading the phenix.autobuild documention correctly, it sounds like there are two modes:

(i) Standard mode - rebuild the whole protein from scratch, which does give the possibility of adding my N-terminal residues. However, I don’t want my nearly complete existing model to be rebuilt, I just want AutoBuild to try to add the missing N-terminal sequence to the existing model.

(ii) Rebuild-in-place, which rebuilds the current model but doesn’t add or subtract any atoms. So that sounds like it’s not what I’m looking for because it won’t add the N-terminal sequence AND it rebuilds the existing model, which I want to keep exactly as is.

Is there some series of options in AutoBuild that will allow me to just add missing residues (provided in the sequence file) but keep all the other currently existing residue in the input model exactly as they are?

Happy to hear any advice you can give.

Thanks,

Matthew

---

*Matthew J. Whitley, Ph.D.*

Research Instructor

Department of Pharmacology & Chemical Biology

University of Pittsburgh School of Medicine

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