Link to this post | posted 20 Jul, 2017 14:26

kklyczek

In addition to small and large terminase subunits, cluster J phages have a third gene with terminase homology. My question is about which gene to call small terminase. The cluster J paper (Pope et al. 2013, doi:10.1371/journal.pone.0069273) identifies the small terminase just upstream of the large terminase (e.g. Baka_5 and 6), and the additional gene (Baka_2) is also identified as a terminase. However, the annotations in Phamerator all have the Baka_2 homologue identified as small terminase, and Baka_5 is not labeled. HHPred results for Klein_2 and Klein_10 (Baka_5 homologue) are attached, showing that Klein_2 has more homology to large terminase and Klein_10 matches small terminase. 2.34Mb

Link to this post | posted 26 Jul, 2017 00:23

washj

Hi Karen, I ran into a similar problem in trying to identify the small terminase in a K2 phage and so I did some digging and found a paper by Shen et al "Functional Identification of the DNA Packaging Terminase from Pseudomonas.." Archives of Virology 2012 Nov. 157 (11) in which they explained that most terminase small subunits have HTH DNA binding motifs but some don't. They used the program COILS2 (http://www.ch.embnet.org/software/COILS_form.html) to identify the small terminase as it reveals the presence of a coiled coil motif (CCM) which is need for protein dimerization. Based on this, I ran both Baka_2 and Baka_5 and it appears that Baka_5 is the small terminase as it has a CCM motif. Baka_2 does not. Jackie

Link to this post | posted 01 Aug, 2017 22:12
dbolliva	Hi Karen, I am just getting started on looking at the J phages, but I noticed that our phage Squint does not appear to have a homolog of Klein_2 but has the homologs of Baka_5 and Baka_6 right next to each other. Just supporting the excellent sleuthing Jackie did. Dave

Link to this post | posted 10 Aug, 2017 18:19
delesall	For the output, are we just looking for the absence or presence of a coiled coil motif?

Link to this post | posted 11 Aug, 2017 21:03

kbutela

Presence of a coiled coil motif. I ran Baka_5 as an example and very clearly saw the CCM Jackie identified (prob. was at least 0.6). Per our discussion on the Cluster A4 terminase genes from yesterday, I ran Blackmoor_2 through COILS2. Although this gene has HHPred hits (87% probability, 53% coverage) to a phage terminase, the probability of a CCM is only about 0.15. I played around with the scoring algorithms recommended in the manual and never got anything better. TinaFeyge_2 is annotated as a terminase, small subunit, but I also didn't get a CCM motif for this gene on COILS2. The evidence is close, but I don't think it meets the threshold to definitively call this gene in the A4s as a small terminase.

Link to this post | posted 11 Aug, 2017 21:27
delesall	So, I should have better phrased my question. Do we want the prob of a CCM to be greater than 0 or greater than as certain value? If so, what is that threshold? prob of 0.2 or 0.5?

Link to this post | posted 11 Aug, 2017 21:39
kbutela	The documentation in the COILS2 help guide suggested a cutoff of 0.5 for a residue to be a part of a coiled coil motif. I may be a bit conservative in my interpretations, but I'm going to stick with the 0.5 cutoff for now. I attached a screenshot of the results I got when I ran Baka_5 through COILS2 using default parameters. 13Kb

Link to this post | posted 16 Aug, 2017 13:50
kklyczek	So for the cluster J phages that have the the Baka_2 homologue, are we calling that terminase (not small or large subunit, just terminase)? Karen

Link to this post | posted 16 Aug, 2017 16:58
dbolliva	I thought we were calling it NKF because it is unlikely to be a terminase?

Link to this post | posted 16 Aug, 2017 17:30
kklyczek	It has strong homology to the N-terminal half of the large terminase subunit, including the ATPase domain. It was identified as a terminase in the cluster J paper, though it may play some other role in phage assembly.