Link to this post | posted 22 May, 2023 15:04

debbie

Hi Susan, Holin is one of those gens that is a delight to call, but difficult. it doesn't surprise me that we were not able to call the holin genes historically. Holin genes are many times now callable because of their pfam hits. In this case, that is exactly true. According to deep TMHMM, there are 3 genes directly upstream of the major tail protein that have more than one transmembrane domains. The first of those - the one I think you are pointing to - is a pfam hit to a holin as you describe. I want to call it a holin and the next 2 transmembrane proteins. I would not be surprised if they too are involved in the holin/lysin function for this phage. debbie Here is paper to help! Edited 22 May, 2023 15:12 962Kb

Link to this post | posted 12 May, 2023 04:34
debbie	Hi Fred and Chris, am not convinced that this isn't something that we could call an HNH. I just skimmed a paper, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592412/ , that seems to suggest that the motif could be HNH, HNK, or HNN. In general, there are a number of endonuclease 'types' that we have not addressed. I am not sure we have a complete enough understanding to differentiate. debbie

Link to this post | posted 11 May, 2023 19:09
debbie	Steve, If you are using DNA maser to submit genomes, you can ignore this. We are in compliance. debbie

Link to this post | posted 09 May, 2023 00:07
debbie	Hi Lee, Yep. that has been happening for a while. I agree it something with the "O", but that is not easily explained. When all else fails, i go to the GenBank page and download a GenBank file and import that way. Dr. Lawrence also knows of this error, but DNA master can no longer be updated. it is inconvenient, for usre. debbie

Link to this post | posted 05 May, 2023 14:44

debbie

Fred, I'm going to provide a general answer to this and then if you need further clarification, please ask. First, I would recommend that we ignore RefSeq at NCBI Blast for now. When we curate and correct our annotations they are not being carried over to the RefSeq data. We do not have control of the RefSeqs so they are there, but I would not use the data. In general, what is ascribed in a GenBank file is someone's interpretation into their investigations for that protein. We continually ask that you find more 'primary' data than that. So use the conserved domain database and the PDB in HHPred. All other pieces of data can again get you to heresay, so carefully evaluate. *And I am attaching the disclaimer that all points here can be broken. As for the specific HNH call, SMART has discovered that when you look at the alignment hits to an HNH, you but have helix-turn helices in your protein, and there must be a conserved H -N -H present. I can validate that we have called HNH's inappropriately in the past and are still rectifying that a bit at a time. Finally, I cannot stress enough to all that using BLAST hits to assign function is fraught with error. And the degree to which it is erroneous is protein dependent. For example, if you blast one the first big genes in the genomes, it is likely the terminase, so when you blast it, and it hits it, you are likely to be right. BUT, when you HHPred it, it will also it a crystal structure of a terminase, so what is the rational of your call. matching what others called OR a significant hit to a crystal structure with all part identified. Terminases are not confusing, so if you stop at a blast hit, you will still be right. But there are other calls that require closer inspection - like HNHs, endonuclease, and endolysins to name a few. There is no catch all direction I can provide for how to navigate this except do the entire bit of work and make the case. Full disclosures, I have not investigated what you asked. So if I have not directly answered your questions, or this would not allow you to correctly assign a function to you your protein, let me know. best, debbie

Link to this post | posted 02 May, 2023 12:52

debbie

Kathleen and students, I would say your phage is canonical for a Cluster A3 phage, and the lysins are where they are and, I guess, where they are supposed to be. And the holin is elusive. I just QUICKLY looked at the proteins with transmembrane and HHPredded gene 30 of BlueBird. It has 2 transmembrane proteins and is located at the end of the minor tail proteins. It has a non-significant hit to a holin. Though I don't reccomened calling it, I would bet a nickel that it is indeed the holin. debbie

Link to this post | posted 02 May, 2023 12:36

debbie

Hi Fred, I think that you have covered all of the bases in this evaluation. I would avoid calling this gene. The instances where I know there is a tRNA in a predicted protein do not look like this one. However, when I saw the hit to an HNH that was confounding. However, I don't think it is an HNH. The H-N-H seems missing to me. I would call call the protein, but include the tRNAs. I would also draw attention to this in your cover sheet. The QCer will review all of this to confirm. Best, debbie

Link to this post | posted 01 May, 2023 17:45
debbie	Thanks!

Link to this post | posted 01 May, 2023 12:56
debbie	Joe and Katie, I am not following this, so I am going to restate. Some people use the function field as a holding area when working in DNA Master. The final minimal file should have nothing in the function field. (All functions are recorded in the Product filed.) In fact, it is good to use the "clear all" buttons in DNA Master to be sure there are no stray characters there. debbie

Link to this post | posted 01 May, 2023 12:51
debbie	Hi Pam, You can submit Hypothetical Protein as Hypothetical Protein or hypothetical protein. The capitalization is related to how it is automated in DNA Master and PECAAN, respectively. Sorry that is confounding, but it is the small stuff. Best, debbie