Link to this post | posted 05 Aug, 2019 22:30

cdshaffer

I always think that an HHPRED of 100% probability is an indication that it is very confident that the protein you are searching with belongs to the same family of proteins it is matching. Chitin is a polymer of β-(1→4)-linkages of N-acetylglucosamine (NAG) while peptidoglycan is alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. I always try to remind my students that functional annotation down to one specific substrate is very risky. In my mind it is easy to change just one or two amino acids at exactly the right place to radically change the binding properties of an enzyme to any one specific substrate. And one should be even more careful when there are known to be many different but related substrates [like polysaccharides]. So annotation of this type needs a "sanity check". Does it make sense that a phage would have an enzyme that cleaves chitin? No, not really, so I would not annotate this as a chitinase. One the other hand, it does make sense that some ancient protein that does something with N-acetylglucosamine could easily evolve into a chitinase along one branch and evolve into some other protein that somehow interacts with peptidoglycan in another branch. I would come away pretty confident that this phage protein either binds to NAG or cleaves β-(1→4)-linkages of NAG. Since there is no approved term for "protein either binds to NAG or cleaves β-(1→4)-linkages of NAG" I would stick with "minor tail protein" but I would also be pretty confident of that annotation given the structural similarity of chitin and peptidoglycan.

Link to this post | posted 19 Jul, 2019 18:05

cdshaffer

Ribosome Modulation Factors (RMF) were originally described as a set of proteins that appear to be involved in ribosome dimerization activity during the stationary phase of bacterial growth. See this JBchem article. According to this uniprot entry they have also been shown to hinder translation initiation. Phage Gilgamesh (see phage in PECAAN for all the details) gene 51 (46895-47104) is a 70 aa long protein which aligns by HHPRED to the v (lowercase v not the capital V) chain of crystal 6H4N and to the A chain of crystal 2JRM. Both these chains were annotated as "Ribosome Modulation Factor". Both HHPRED alignments are in the high 80-90% coverage, 99% probability and an e scores of around e-11. HHpred also aligns to the Pfam entry for ribosome modulation factor, with about the same coverage and e values. I see no reason to modify the current term used and would stick with "Ribosome Modulation Factor" and it is generic enough to cover how phage might be using these proteins differently than the host but the function is still likely to have something to do with modulating ribosome activity. Edited 19 Jul, 2019 19:57

Link to this post | posted 15 Jul, 2019 20:07

cdshaffer

for phage Saftant gene 68 (45610-45200) we have found good evidence for an anti-restriction protein but the matches are not to ArdA-like like proteins but rather a second type of antirestriction proteins called OCR. The OCR-type anti-restiction protein is found in phage T7 and is one of the first protein products expressed following infection (see this NAR paper). OCR type are typically shorter (~120 AA) than ArdA (170-180) and have sufficiently different crystal structures that I think we should avoid using the ArdA-like approved term for this protein. Here is the image of the two structures side by side. For details, see phage saftant in PECAAN but briefly saftant 68 hits by hhpred with 94.4% probability to the crystal of gene 0.3 protein from phage T7 (D chain of this crystal) with ~90% coverage of both subject and query. I would propose either adding this second type as an approved function "antirestriction protein, OCR-like" or simply adding another term without the ArdA-like (i.e. "antirestriction protein" ) to avoid over annotation. Edited 15 Jul, 2019 20:10

Link to this post | posted 28 Jun, 2019 19:11
cdshaffer	Christian and I are working on a version of the VM with the experimental version of starterator in the faculty account as well as k-phamerate for validation and phamnexus for splitstree prep. Unfortunately, there is no easy way to get the newer version of aragorn that trims the genes properly installed on that old Ubuntu 14.04 VM.

Link to this post | posted 25 Jun, 2019 18:23
cdshaffer	OK since we are working on a few phage here, I have installed command line Aragorn ver 1.2.38. If anyone is in a hurry and does not want to wait until web based Aragorn is back up, I can easily run aragorn command line for you, just send me an email with the sequence as a fasta file attachment and I can email back the aragorn results.

Link to this post | posted 25 Jun, 2019 16:33

cdshaffer

A quick google search suggests that the "ERR_CONTENT_LENGTH_MISMATCH" error is probably an issue with nginx (the web server software) so yes it is time for an email. I did look for anywhere else that was running public available Aragorn but only found a galaxy server running the older 1.2.36 which does not appear to trim correctly (based on a single prediction in phage saftant that we ran to test). If anyone is desperate, one can easily install the command line version with conda on mac, or apt-get on ubuntu version 18 or 19. Still looking for a viable web based solution. Edited 25 Jun, 2019 17:44

Link to this post | posted 05 Jun, 2019 18:04

cdshaffer

In situations like this we are left with the very hand wavy answer that it is simply that the functions provided by the holins are being provided by proteins which cannot be detected with bioinformatic based tests. This can happen by either (1) convergent evolution (i.e. in this context, two completely different proteins evolving to carry out the function) or that (2) a completely different biochemical pathway has evolved in the B1 phages to "solve" the problem of how to lyse the host cell wall In either situation bioinformatics fails us (by definition really) and we are left going back to the wet bench to answer the question "how do B1 phage lyse their host?" So maybe someone else might be aware of published or unpublished results from bench work in this area and might have further insights.

Link to this post | posted 28 May, 2019 20:12

cdshaffer

I have just returned from vacation and have updated the the starterator pages to the most recent version of the phamerator database which was posted this morning. I checked bunnies gene 32, it is now in pham 39047 and appears to be in sync across pecaan, starterator, phagesdb.org and phamerator.org. Checking phage Fulbright shows some discrepancies across the various platforms but that could be syncronization issues since the last update was posted so recently. I would give it a day and if reports are still missing please repost and I can look into it.

Link to this post | posted 10 May, 2019 21:57

cdshaffer

Just a heads up that I will be out of the country for the next two weeks and will have unknown quality of internet connections throughout my travels. As such, it is likely I will not be able to update online starterator reports in a quick and timely fashion. Most pham groups remain static and the old report should suffice in most cases. However, should an updated version of the pham database move your gene of interest to a new pham number the virtual machine remains a solution if you are in a hurry. See this page for help. Thank you for your patience and understanding. Edited 10 May, 2019 21:59

Link to this post | posted 10 May, 2019 21:44
cdshaffer	there is a button that should do that but if the button on each gene is not working the whole phage might not either. Go to Admin menu -> phages; search for your phage and click the edit button. From there you should get a page which includes button to reblast the whole phage.