Apologies, this should have been directed to the mailing list. Gabor -------- Original Message -------- Subject: Re: [phenixbb] Scultor model with gaps included Date: 2016-05-25 14:33 From: Gabor Bunkoczi To: Joseph Brock Dear Joseph, apologies for the late response, and sorry for the trouble. Please find some answers inlined. On 2016-05-24 15:10, Joseph Brock wrote:

Dear Phenix community,

I was hoping if someone could help me with using Phaser correctly, since I can't seem to produce the desired search model.

The first thing that I was wondering about is that the CLUSTAL alignment from the log file is quite different to the .aln alignment I made with phenix --> other tools --> multiple sequence alignment. Why is a sequence alignment required as input if sculptor performs it's own internally?

Sculptor can perform sequence alignment, but the algorithm is relatively basic, and you can get much better results with an external method (such as HHPRED), and hence alignment input is preferred over the target sequence. The alignment selected is printed into the logfile for reference. On the other hand, Sculptor does align the structure of your model chain to the sequence of your model (this is a surprisingly non-trivial task). However, this alignment should be close to 100% identical, unless a mistake has been made (such as using the wrong alignment file). You should get a warning if the sequence identity is too low. It is normally OK to have a couple of mismatches (such as having SeMet in the model structure with translates to "X" and not as "C").

My model sequence aligned to my target sequence in the log file has several gaps in the alignment . For e.g.,

MASTER ......AASFIMV...... CHAIN_A ......AKM--MF.......

I would like to great a search model in which the residues aligned to gaps in the MASTER chain are included, however the resulting sculptor .pdb file has these residues removed. Shouldn't they be inserted so that the rest of the chain is in the correct register?

You need to do homology modelling to achieve this, as an insertion can affect the whole structure. Sculptor is designed for quick model improvement, and just renumbers/renames residues. However, the new residues numbers should reflect your sequence, i.e. in your example alignment the two methionines on both sides of the gap will get a non-consecutive number and are displayed as non-bonded in e.g. Coot.

If anyone can let me know the parameters to allow this, I would greatly appreciate it. I tried unselecting 'gap' in main-chain deletion scope and some other variations, but this didn't seem to help.

I seem to get the same result even if I change the target index from 1 to 2 in the associated .aln alignment. Does anyone know why this is?

Hmm, this does not sound right. Can you send me the logfile so that I can have a look (please use my direct email address)? Best wishes, Gabor

Apologies if this is quite obvious, I have been reading the documentation but can't seem to figure it out.

All the best,

Joseph.

Joseph Brock | PhD Division of Physiological Chemistry II Department of Medical Biochemistry and Biophysics Karolinska Institutet Scheeles väg 2 SE-171 77 Stockholm, Sweden

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