Link to this post | posted 26 Jun, 2020 18:47

anders

Hi Welkin, Yes, when I remove the inserted HNH and the 6 bp duplication that was created, a lysin A is made that is identical to others such as MilleniumForce lysin A. I've attached my notes and sequences. Thanks, Kirk ###################################################### addendum: I have also attached documentation of the final decision we made for the splice junctions. Edited 08 May, 2021 00:54 522Kb 154Kb

Link to this post | posted 23 Jun, 2020 06:27

anders

Hi, I am QCing the cluster F phage Coco12, which has an HNH endonuclease inserted into lysin A, and am looking for guidance on annotating the lysin A bits. The insertion seems to split the gene between domains. HHPred easily identifies the amidase domain in the downstream half, but it does not provide any evidence for a peptidase domain in the upstream half. BLAST does show that the upstream half has the same sequence as lysin As in Payne and Hatfull that contain the "N3" putative peptidase domain, as in Tweety gp30 (Payne and Hatfull, 2012). Cluster F phage Frankie has nearly the same situation with an HNH endonuclease inserted in nearly the same position. To my surprise, the lysin A gene in Frankie is annotated as if it has an intron – in two sections that are connected together to make one lysin A polypeptide. The GenBank entry reads gene 26909..28665 /locus_tag="SEA_FRANKIE_33" CDS join(26909..27370,27946..28665) I have attached phamerator screenshots of the Coco12 and Frankie lysin As aligned with intact lysin As. Should Coco12 lysin A be annotated with an intron, as in Frankie? (I am thinking that bench evidence ought to support an annotation like this.) Or, should each lysin A part be annotated with a domain function appended? We have good HHPred evidence supporting the amidase domain for the downstream section, but we lack HHPRed or CDD evidence for a peptidase function in the upstream bit. BLASTp shows that the upstream section is nearly identical to Tweety gp30, which is the canonical lysin A listed in Payne and Hatfull that carries the N3 hydrolase domain (M23B peptidase in phi29 gp13 tail protein). Or is there another option that is the best in this case? Thanks! Kirk ################################################# Addendum 6/26/20: I said above that the first half of Coco12's lysin A did not hit anything on HHPRed. I have never fiddled with HHPred settings before, but I changed the MSA generation method from the default HHBlits=>UniRef30 to PSI-BLAST=>nr70 and this now hits a group of proteins that does include phi29 gp13. The region that matches phi29 gp13 is not to its m23 metallo-peptidase-like domain, but to its lysozyme domain. I've attached a screenshot and link to the HHPred output. Edited 26 Jun, 2020 16:49 150Kb 101Kb

Link to this post | posted 28 Jun, 2018 17:47
anders	The evidence for a non-syntenic head-to-tail connector is weak (HHPred < 90% and matches only 1/2 of gp6/HK97). Kerry (gene 11) and Gravy (gene 11) and singleton Camille (gene 8 ) should probably be changed to NKF.

Link to this post | posted 28 Jun, 2018 17:41
anders	thanks, Welkin. I will post an annotation tip in Cluster DJ.

Link to this post | posted 28 Jun, 2018 00:51

anders

A gene next to lysinA and holin has been annotated as head-to-tail connector complex protein in the singleton Camille (gene 8 ) and Gordonia DJ phages Kerry (gene 11) and Gravy (gene 11). 33 other pham members have been annotated NKF, and one as structural protein. Two things make me wonder about the evidence for this function, abnormal synteny and a small fraction of the protein matching by HHPred. 1. In DJ genomes, this gene is not in-between capsid and major tail genes, as they usually are. It is upstream of capsid and is downstream of lysinA and holin. Is this a feature of Gordonia phages? 2. When I look at the HHPred evidence, I only see two alpha helices in these proteins matching to gp6 of HK97, which is a small fraction of the DJ protein and a small fraction of gp6-HK97. HHPred predictions are 40-75%, less than 90%. I have attached some screenshots of the HHPred output and the region of gp6 on PDB. Are there other pieces of evidence that makes this function call stronger? Edited 28 Jun, 2018 00:52 242Kb

Link to this post | posted 07 Aug, 2017 22:05
anders	As called in Gordonia phage Bantam gene 3. The protein I'm looking at is a homolog, Phabba_draft_263. This is called a number of things; PRPP synthetase is common. https://toolkit.tuebingen.mpg.de/#/jobs/4777687

Link to this post | posted 07 Aug, 2017 21:25

anders

I'm working on Phabba, the only other C2 besides Myrna. In the region that is at the same spot as the lysis cassette in C1, there are three genes. The first has good homology and HHPred structure to endolysins and phage genes called lysin A. The second gene has good HHPred structure matches to glycosyl hydrolases, Beta-xylosidase/alpha-L-arabinfuranosidases. The third gene has two predicted trans-membrane domains. I have a phamerator screenshot at https://docs.google.com/document/d/1kon45w_z70hj0j5hVxiL4Os4_teCo3pEnfWDxFEFNh4/edit?usp=sharing I can confidently annotate the second and third as "hydrolase" and "membrane protein". Qs: 1. What features define lysin B? Could this glycosyl hydrolase be a lysin B? 2. Should this membrane protein be annotated as a holin since it is near to a lysin A? thanks, Kirk

Link to this post | posted 07 Aug, 2017 04:58
anders	For such enzymes that are not in the RuvC, RusA, or endoVII families of Holliday junction resolvases. Please see Lee's and my discussion in the Function calling section. Thanks :-)

Link to this post | posted 07 Aug, 2017 04:52

anders

I've got a gene in a C2 that is like this. The HHPred hits are to an archeal Holliday junction resolvase. When I use RuvC or RusA in HHPred, these do not hit this class of Holliday junction resolvases. Here is a paper reports the structure of the top hit: https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkh869. In the introduction, it says that RuvC resolvase and RusA resolvase (from bacteria), and Endonuclease VII (from T4) are also Holliday junction resolvases. All of them are members of the type II endonuclease superfamily. This archael Hj resolvase is similar in structure to the HJ resolvase in Bacillus, RecU. If I understand this right, I feel like if I assign RusA or RuvC or endoVII to this protein, it would certainly indicate that it is a protein that resolves Holliday junctions, but it would not be accurately place the structure into the right structure family. The protein is Phabba_draft_198. These are the HHPred results: https://toolkit.tuebingen.mpg.de/#/jobs/4369808 (available for about 2 weeks, I think). Turns out that Phabba_draft_202 also hits these same structures in HHPred, although it doesn't have an obvious AA match to gp198. 202 hhpred: https://toolkit.tuebingen.mpg.de/#/jobs/9936992 Holliday junction resolvase is an unofficial function that has been assigned to some of its homologs in other phages. Maybe we could have this be an official function and use it when it's not RuvC, RusA, or endoVII. Alternatively, maybe we could name this one, too. HJC Holliday junction cleavage? RecU Holliday junction resolvase? I will make a post in the New functions request area. Edited 07 Aug, 2017 05:08

Link to this post | posted 06 Aug, 2017 21:26

anders

Phabba_draft_83 HHPred hits many acetyltransferases. The matches are across most of the acetyltransferase domain in each alignment. This link to HHPred results should be live for about 2 weeks: https://toolkit.tuebingen.mpg.de/#/jobs/6362589. I'm attaching the full HHPred output as text. With aa sequence alignments, the alignment is narrowed to just the central acetyl-CoA binding portion on CDD. Edited 06 Aug, 2017 21:30 187Kb