Link to this post | posted 15 Dec, 2020 18:00

cdshaffer

OK this appears to be an out of sync error. The current version of the database is 387. The starterator analysis to create the 14 thousand or so reports is on going so the web pages still report the old version 386. The computations should complete and be posted by the end of the day at which point the links from pecaan and phagesdb should work. In the mean time you can use these links to get to the reports for those three genes in the older 386 database reports here: Peel 30: old pham number 44662 and link http://phages.wustl.edu/386/Pham44662Report.pdf Peel 32:old pham number 44868 and link http://phages.wustl.edu/386/Pham44868Report.pdf Peel 33:old pham number 44899 and link http://phages.wustl.edu/386/Pham44899Report.pdf There is a way to check if the "out of sync" error is likely to be the cause of these types of missing phams by comparing the reported database versions. This link will always show the current version number on the Starterator server. Alternatively, you can look on any starterator report where you will see something like this: "This analysis was run 12/09/20 on database version 386" near the top of the first page of text. In pecaan you can go to the Pham Maps page and look just above the map where you will see something like this: Phamerator Version: 387 . Unfortunately there is no easy way to get the version number of the database at phagesdb but you can always check this link to see which version is on the database server, all the programs download from there. Edited 15 Dec, 2020 20:47

Link to this post | posted 15 Dec, 2020 16:52
cdshaffer	These are typically due to database at phagesdb being out of sync with the current database for the starterator reports but this should not be happening "for weeks". Are you using pecaan or phagesdb for your links? Could you post the pham numbers you are looking for?

Link to this post | posted 10 Dec, 2020 18:58
cdshaffer	Ok, those look pretty good. I now suspect our issue might have been with the age/storage of the grids. Thanks

Link to this post | posted 10 Dec, 2020 16:45

cdshaffer

Our dept purchased an SEM with a TEM detector a couple of years ago. We tried several times but were never able to get sufficient resolution to see phage on the grid. To be fair, we were just testing older grids that had been returned after imaging at the EM core so fresh grids might have given us viable images; but it really looked like we were never going to get the resolution we needed to see things as small as phage. If you can get it to work please post your protocols we would be very interested, it would open up some interesting curriculum opportunities. Sorry I don't know any of the specifics on our system but I could ask our microscope facility if you want the specifics. Edited 10 Dec, 2020 16:49

Link to this post | posted 09 Dec, 2020 22:55

cdshaffer

For those of you using Phamerator and Starterator in the SEA VM: The Hatfull lab has been doing some much needed updates to the underlying phamerator database and at the same time has moved all the databases to a new server. The combination of these two events means that the SEA VM version of phamerator and starterator will no longer be able to update to new databases as they are released. These programs will continue to work "as is" but the databases they have now will become increasingly out of date as new phage are added and pham assignments invariable change. The production version of starterator has been updated and the web based PDF's are up to date with the new database. Users of the web pages should be on the lookout for any discrepancies or possible errors, I have tested many pages but cannot screen all 14,000 or so pham reports so it is possible that some unusual set of circumstances could result in errors in the Starterator reports. If you find anything of concern please report to this forum or send me an email directly. Finally, I am looking for a few volunteer testers to work with me on testing the new beta version of Starterator in their VM. Send me an email if you would like to help in this regard. Thanks. Edited 09 Dec, 2020 22:59

Link to this post | posted 07 Dec, 2020 19:10
cdshaffer	Thanks for posting this. I have added a note on the issues tracker for Starterator here: https://github.com/SEA-PHAGES/starterator/issues/42 Hopefully once the end of the semester grading bonanza ends I can update starterator to handle the new default Genome profile format created by DNA Master.

Link to this post | posted 25 Nov, 2020 00:26

cdshaffer

These issues of specificity are always difficult to annotate. I agree with Debbie in that I tend to "do no harm"; that is, I would annotate using the more general "protease" unless there is good evidence for the more specific term. To answer the question of function at this level you would have to dig into the published literature on the matching crystals and see if they describe the active site in detail. If you are lucky you can find enough detail as to exactly which bonds are involved in the reaction and which side chains are critical in the active site. If you can get that, then you could see if your protein is likely to create that same active site using 3D modeling and visualization tools. This is well beyond what I ask students to do in general but we ask students to investigate one gene "in depth" and this would be a really good candidate gene for this kind of detailed investigation. The likelihood of success is low, and I tell my students that up front, as they have to be lucky enough that the information they are looking for is actually in the published literature. But as a teacher I am fine with students trying as it is really about the journey not the destination. Edited 25 Nov, 2020 00:29

Link to this post | posted 18 Nov, 2020 23:42

cdshaffer

In phage Belfort CDS 134(87,804-88,487) has a large number of high quality hits to NAD-Dependent Deacetylase. There are approx 50 HHPRED hits with 100% probability and >99% aligned. The vast majority of the top hits include the term "sirtuin" a group of enzymes found in all kingdoms. However, of the 100% probability alignments more than half include the term "NAD-Dependent Deacetylase". The top prokaryote hit is to crystal 1S5P_A, an enzyme from Escherichia coli (100% probability and 99.5% coverage) and has the description "NAD-dependent deacetylase (E.C.3.5.1.-); protein deacetylase". We propose either the term "NAD-dependent deacetylase" or "NAD-dependent protein deacetylase" and avoid the whole "sirtuin" nomenclature. If you want to see all the hits, this phage is in PECAAN (Belfort 134). For detailed alignments here is the amino acid sequence to rerun the HHPRED search: >Belfort_cds_134 MVKVLFVTGAGISANAGIPTYRDGGSSWKDADLEKKSHASRYGNHLDELWDKHWGPVAKAMGQAEPTQTHRAIAEF QKDNPSIVATQNIDDLHERAGSDNVAHVHGSMVIKCIRCKRSHLETKWFGKGAPVCPHCGKSKTRPDVVLFGEKLD LKMFAALESFAKHDADVIVAVGTSLNVFPAAGLVMDNIAKSVIINKEKTPFDKFACKVYNDDCDSVIDEVLGGLN

Link to this post | posted 22 Oct, 2020 00:15

cdshaffer

To me the results of of the starterator reports are quite telling. The two choices you point out are labelled start 12 and 15 in the current starterator report here. First the level of conservation for start 12 is much much higher than start 15. In fact there are only 2 of 56 phage that don't have start 2 and both of those have a start very very close by position to start 12. On the other hand start 15 is only seen in 2/3rds of these genes and for 7 of the 30 tracks there are no starts anywhere near start 15. To me it is hard to believe that evolution would continue to choose to keep the bases that code for start 12 in virtually all these genes if start 15 was really the start cf translation, so I would have a strong preference for it. As for coding potential (CP). If you look carefully you can see examples of other regions in the genome where you know the sequence is coding but the CP signal drops to zero. These are regions that are downstream of a strong CP signal but before the stop codon. See the CP for gene 14, there is easily at least 100 bases with no CP signal. So this is why I have a "rule" that a positive signal in CP is good evidence there IS a gene but no CP is not quite as good at indicating there IS NOT a gene. Said more formally, CP algorithm makes more false negative errors than false positive errors. So, in this case where one start says there is a CP false positive (start with 245 gap) and the other choice would say that CP is a false negative I would say that CP also is slightly more supportive of the big overlap start. Taken together then I would annotate this gene to start at 1322. If I were helping a student with this I would now ask them to back and double check that gene 2 is real just because of that super large overlap. But even if gene 2 is real I would probably still stick with that huge overlap given the strong level of conservation seen in starterator report.

Link to this post | posted 24 Sep, 2020 20:17

cdshaffer

The term "AAA-ATPase" is a domain found in a wide variety of proteins and is indicative of a specific fold pattern that creates an ATPase pocket that has been seen in many different proteins. So an annotation with that term says you want people to know that the protein very likely cleaves ATP and uses that specific fold structure. The term "terminase" reflects the biological role the protein supplies for the phage. To me, neither term is inherently better than the other, it all depends on what one is interested in. I am sure there are many enzymologists out there that are much more interested in the presence of an enzymatic domain, while others are more interested in why the phage would have that gene at all. My own perspective is to go with the biological role if I can find sufficient evidence in support and only mention the presence of a domains (as they are often good hints to the biological role) when there is not sufficient evidence to call the role.