Link to this post | posted 09 Jan, 2018 18:06
cdshaffer	If you are getting the access violation when trying to auto-annotate check out the DNA Master troubleshooting guide here: http://phagesdb.org/DNAMaster/faq/ If it is happening somewhere else, could you give more details on exactly which steps you are taking and what you did just before the got the error.

Link to this post | posted 15 Dec, 2017 20:40

cdshaffer

Analysis of protein OzzyJ_38 draft annotation. When we run an HHPRED analysis at the original site, the top hit is to a crystal structure with 99.18% probability and an e-score of 2.6x10-12 to pdb crystal structure 2WCW which is described as a Holiday junction resolvase from Archaeoglobus fulgidus. At this point looking into the issue of it was a "RusA" or "RuvC" type of resolvase we looked at the 4th best HHPRED hit (which actually had an associated published paper) with a probability of 98.81% and e of 4.9x10-10 to the pdb crystal structure for 1GEF. This is also described as a Holiday junction resolvase but was from the hyperthermophilic archaeon Pyrococcus furiosus. The paper describing these results (Structure. 2001 Mar 7;9(3):197-204) describes this structure as "The folding of the Hjc subunit is clearly different from any other Holliday junction resolvases thus far known." Instead the overall structure is described to be similar to type II restriction endonucleases. For now, we have submitted this protein with the simple annotation of "holliday junction resolvase" since it is neither a RusA or RuvC type. However, if adding a type was preferred, adding a descriptor like "Archaeal" or some indication of its similarity to type II RE enzymes might be appropriate. Follow on thoughts appreciated.

Link to this post | posted 07 Dec, 2017 16:32

cdshaffer

Chelsea, As a back up plan students can enter their data directly at phagesdb.org. Ideally students should upload EM, digest, and plaque pictures (if they have them). To prepare images for upload see this: http://phagesdb.org/media/workflow/protocols/pdfs/MakingThumbnails.pdf Also, before going to uploading everything, go get the GPS coordinates using Google maps. Just go to google maps and zoom in to the point where you can accurately click on the collection site, after clicking exactly on the spot in the map a box will show up with the decimal GPC coordinates, just copy them for the last step. Go to the add a phage page on phagesdb.org which is here: http://phagesdb.org/addphage/ Or if the phage is already been added to phagesdb.org they can go here to make updates: http://phagesdb.org/modphage/ Students should fill out as much as they can but some fields will certainly be left empty as they require the genome sequence. I especially encourage my students to fill out the Naming notes section; mostly because I am always curious the how and why of a particular phage name.

Link to this post | posted 29 Nov, 2017 16:45

cdshaffer

I think this issue of the phamerator database synchronization is the most pernicious problem out there. I have been trying to think of ways to improve the system but it is tricky given the various distributed programs that use the database. I keep some older versions of the database just for historical records. It looks like the last version of the database with pham 18287 was version 117. You can see the starterator report here (note the 117 in the URL: http://phages.wustl.edu/117/Pham18287Report.pdf That pham is not found in my next stored version (120), with that version the gene Kareem_11 has moved to pham 29797: http://phages.wustl.edu/120/Pham18287Report.pdf When using the SEA VM the only way to update to the most recent version of the phage database is to run phamerator. Once you start phamerator it will check in to see if there is an update, and if so, it will attempt to download and install. Once you get a successful database update then the SEA VM Starterator will match the current online versions.

Link to this post | posted 14 Nov, 2017 00:12

cdshaffer

This is a proposed funtion based on Pham 28735 (example member Mildred21 gp255) The HHPred has a 100.0%/e=9E-39 93% length alignment to an NMR structure to e coli YibA in PDB and to COG3236 see screen capture of hhpred alignment here. The phyre2 3d prediction is a 100% confidence with 91% coverage to the same pdb structure. This link should work for about a week. here is the screen capture which should work for the foreseeable future. YibA is a founding member of COG3263, the function for COG3263 is based mostly on this paper: A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis. Biochem. J. 2015 Feb 15; 466(1):137-145 This paper clearly shows that this e coli protein (and a couple of others like it) can enzymatically function to cleave N-glycosidic bonds in vitro as part of a large complex that carries out the first few steps of riboflavin biosynthesis. So there is really good evidence for enzymes of this type to be able to hydrolyse N-glycosidic bonds. I am certainly not proposing that this enzyme in phage is involved in riboflavin biosynthesis, but the wet bench is pretty good that the e coli YibA does have a glycosylase activity and there are many different glycosylases. The issue as to naming is one of specificity, should we use the more specific N-glycosylase or the more generic glycosylase? There are also S, O and C- type glycosylases. I feel that without biochemical evidence or a published analysis of the amino acids present at the active site and how they interact with a particular glycosidic bonds (i.e. how does the active site change with N, S, C and O type glycosidic bonds) that specifically adding the term N-glycoslyase may be too specific given the evidence above is simply bioinformatic alignments. Following this logic, I would say the more general glycosylase term is preferable. On the other hand, of all the glycosylases, one might argue that the most likely activity for a phage enzyme is as a DNA glycosylase. All of the DNA glycosylases are of course N-glycosylases (i.e. all 4 bases connect to the ribose through a nitrogen). I think the evidence for this phage protein family to be an enzyme is about as good as you can get with just bioinformatic alignment so adding something is probably appropriate, the question is how specific do we want to go, the generalized glycosylase or the more specific N-glycosylase.

Link to this post | posted 09 Nov, 2017 20:42
cdshaffer	What is the recommended protocol for suggesting updates to the newly posted list? For now I will just suggest here that we add "dNMP kinase" to the "do NOT Use" column for the written out entry "deoxynucleoside monophosphate kinase".

Link to this post | posted 31 Oct, 2017 14:51
cdshaffer	Wait, there is a new list posted? I looked at seaphages.org under faculty info and phagesdb.org under workflow/annotations. Both those are the Nov 2015 version. Link please. Edited 31 Oct, 2017 14:53

Link to this post | posted 30 Oct, 2017 15:23

cdshaffer

I am not sure if this is the same issue since I did not see the exact error message but I had a student with a failed install. It turned out he had a high end "gaming" laptop which had two hard drives. A small fast SSD drive which was the "C:" drive, and a slower much larger "D:" drive. He chose to install on the D: drive and was never able to get DNA Master to work properly. It did work when he removed DNA Master and then ran an install on the C: drive. You might want to check with the student and see if he also had two hard drives and if so be sure to install on the C: drive.

Link to this post | posted 19 Oct, 2017 16:13
cdshaffer	If you are getting the "Database setup required" window that looks like this. you just need to enter the root database password "phage" (without the quotes) to authorize updating the system. If you are getting a different kind of password error box can you send the exact wording or a pic. Edited 19 Oct, 2017 16:18

Link to this post | posted 03 Oct, 2017 21:12

cdshaffer

As a follow up. I just tried running auto-annotation on DNA Master and got yet a different set of genes from what is on phagesdb. So my suggestion above will not actually help. I don't know where the discrepancies are coming from, but again likely from using different installations of glimmer/genemark to get the auto-annotation gene lists. The only way for you to get your DNA Master file in sync with the starterator/phagesdb would be to edit the DNA Master file by adding and deleting genes until the list matches up. We will have to wait and hear from Dan on why the phagesdb gene list does mot match the DNA Master auto-annotation.