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Link to this post \| posted 03 Jul, 2018 19:54
cdshaffer	Phage Justbecuase (cluster BM, available on pecaan) gene 252 starting at 149782 has a more than 50 HHPRED hits all with >99% probability and 85%-95% coverage with a variety of descriptions. Examples include "RNase III inhibitor", "Appr-1-p processing domain", "O-acetyl-ADP-ribose deacetylase", and most commonly something about "Macrodomain protein". It turns out there is a large body of literature on "macrodomain" proteins which have been shown to be involved in a wide variety of biological functions across all kingdoms (this wikipedia article is pretty good at summarizing). Interestingly, one of the well characterized prokaryotic uses of this domain has been shown to be involved a toxin/antitoxin system that use UDP-riboylation of DNA. This domain was found and annotated previously in phage Trina gene 253. Here full annotation in the genbank file is "ADP ribose-1-P processing protein, antitoxin" while phagesdb appears to drop the last part and just has "ADP ribose-1-P processing protein". Looking over quite a few of the published structures and enzymatic functions of many of the HHPREd hits, the common thread appears to be enzymes that break/create bonds between an ADP-ribose moiety and something else, be that a protein (see the PARPS), a nucleic acid (as in the DART/G toxin anti-toxin), a phosphate group (in the APPR1-p processing proteins), nicotinamide (see the NAD(+) ADP-ribosyltransferases) , or an acetyl group (see the O-acetyl-ADP-ribose deacetylases). I really don't like the generic term "macro domain" even though most of the literature uses that term. Instead I would propose the term "ADP-ribosyltransferase" which I think does a good job of summarizing all the different activities that have been associated with various macrodomain proteins. While the toxin/antitoxin angle is intriguing I am not sure there is sufficient evidence to support including this biological role as part of the annotation without supporting evidence from something other than HHPRED or BLAST alignments.

Link to this post | posted 03 Jul, 2018 19:54

Phage Justbecuase (cluster BM, available on pecaan) gene 252 starting at 149782 has a more than 50 HHPRED hits all with >99% probability and 85%-95% coverage with a variety of descriptions. Examples include "RNase III inhibitor", "Appr-1-p processing domain", "O-acetyl-ADP-ribose deacetylase", and most commonly something about "Macrodomain protein".

It turns out there is a large body of literature on "macrodomain" proteins which have been shown to be involved in a wide variety of biological functions across all kingdoms (this wikipedia article is pretty good at summarizing).

Interestingly, one of the well characterized prokaryotic uses of this domain has been shown to be involved a toxin/antitoxin system that use UDP-riboylation of DNA.

This domain was found and annotated previously in phage Trina gene 253. Here full annotation in the genbank file is "ADP ribose-1-P processing protein, antitoxin" while phagesdb appears to drop the last part and just has "ADP ribose-1-P processing protein".

Looking over quite a few of the published structures and enzymatic functions of many of the HHPREd hits, the common thread appears to be enzymes that break/create bonds between an ADP-ribose moiety and something else, be that a protein (see the PARPS), a nucleic acid (as in the DART/G toxin anti-toxin), a phosphate group (in the APPR1-p processing proteins), nicotinamide (see the NAD(+) ADP-ribosyltransferases) , or an acetyl group (see the O-acetyl-ADP-ribose deacetylases).

I really don't like the generic term "macro domain" even though most of the literature uses that term. Instead I would propose the term "ADP-ribosyltransferase" which I think does a good job of summarizing all the different activities that have been associated with various macrodomain proteins. While the toxin/antitoxin angle is intriguing I am not sure there is sufficient evidence to support including this biological role as part of the annotation without supporting evidence from something other than HHPRED or BLAST alignments.

Posted in: Request a new function on the SEA-PHAGES official list → justbecause 252 @ 149782 "ADP-ribosyltransferase"

Link to this post \| posted 29 Jun, 2018 17:22
cdshaffer	Online aragorn call the reverse strand tRNA from 88542-88627 as a Pyl amino acid and an anti-codon of cta (which pairs with the amber stop codon UAG). Pyl (pyrrolysine) is one of the noncanonical amino acids which have been described but are fairly rare. A pubmed search of Pyrrolysine in streptomyces only found examples of using a system cloned out of an archaea into streptomyces for use in amber supression. online tRNA scan calls this with an infernal score of 55.7, type undet and anti-codon of NNN. So, are we calling these specialized amino acids and anti-codons if called by Aragorn 1.2.38? Or should we go to a less specific designation of a tRNA with unknown aa and anti-codon? Edited 29 Jun, 2018 20:46

Link to this post | posted 29 Jun, 2018 17:22

cdshaffer

Online aragorn call the reverse strand tRNA from 88542-88627 as a Pyl amino acid and an anti-codon of cta (which pairs with the amber stop codon UAG). Pyl (pyrrolysine) is one of the noncanonical amino acids which have been described but are fairly rare. A pubmed search of Pyrrolysine in streptomyces only found examples of using a system cloned out of an archaea into streptomyces for use in amber supression. online tRNA scan calls this with an infernal score of 55.7, type undet and anti-codon of NNN.

So, are we calling these specialized amino acids and anti-codons if called by Aragorn 1.2.38? Or should we go to a less specific designation of a tRNA with unknown aa and anti-codon?

Edited 29 Jun, 2018 20:46

Posted in: tRNAs → phage justbecause tRNA @ 88542

Link to this post \| posted 29 Jun, 2018 15:12
cdshaffer	We have a gene in the BM streptomyces phage Satis [gp211 with annotated start @129464], with around 40 HHPRED hits with > 99% prob and > 98% coverage with short descriptions of "cold shock protein" and one hit of similar quality to an "RNA chaperone". Investigation into these cold shock proteins suggest they also act as chaperones. See this JBC paper. I think either a generic "chaperone" or a more specific "RNA chaperone" would summarize the results here. For the recent submission we took the more conservative route of just "chaperone".

Link to this post | posted 29 Jun, 2018 15:12

cdshaffer

We have a gene in the BM streptomyces phage Satis [gp211 with annotated start @129464], with around 40 HHPRED hits with > 99% prob and > 98% coverage with short descriptions of "cold shock protein" and one hit of similar quality to an "RNA chaperone". Investigation into these cold shock proteins suggest they also act as chaperones. See this JBC paper.

I think either a generic "chaperone" or a more specific "RNA chaperone" would summarize the results here. For the recent submission we took the more conservative route of just "chaperone".

Posted in: Request a new function on the SEA-PHAGES official list → Satis 211 @129464 "chaperone"

Link to this post \| posted 22 Jun, 2018 16:45
cdshaffer	Yes, you are correct, in every case I have worked with lately Pecaan is giving the codon in the downloaded notes (i.e the reverse complement of the anti-codon reported by tRNA-scan). I always change the final notes back to the anti-codon before creating the genbank submission file. Since Pecaan reports the codons in uppercase, I make the anti-codons lower case which helps me keep track if that step has been completed and matches the examples from the online documentation. In genbank the "tRNA-Thr(ggt)" info ends up in the "Notes" field. There is no official standard for that field in the official documentation here. So I think the important thing is to have everything from the SEA-Phages use the policy as stated in the online docs linked above.

Link to this post | posted 22 Jun, 2018 16:45

cdshaffer

Yes, you are correct, in every case I have worked with lately Pecaan is giving the codon in the downloaded notes (i.e the reverse complement of the anti-codon reported by tRNA-scan). I always change the final notes back to the anti-codon before creating the genbank submission file. Since Pecaan reports the codons in uppercase, I make the anti-codons lower case which helps me keep track if that step has been completed and matches the examples from the online documentation.

In genbank the "tRNA-Thr(ggt)" info ends up in the "Notes" field. There is no official standard for that field in the official documentation here. So I think the important thing is to have everything from the SEA-Phages use the policy as stated in the online docs linked above.

Posted in: PECAAN → PECAAN and tRNA notes problem?

Link to this post \| posted 18 Jun, 2018 15:17
cdshaffer	I thought the same thing but no luck. The intein is entirely within the second (in order of transcription) gene and the upstream gene appears to no longer be expressed.

Posted in: Functional Annotation → fushigi_draft 52 homing endonuclease + primase

Link to this post \| posted 18 Jun, 2018 02:55
cdshaffer	Not sure exactly how to annotate this gene from fushigi (37,851-36310 rev) most A1 phage in this region have the two DNA primase genes with the large overlap, however in fushigi and a few others there is a single large gene of 513 amino acids: The first 128 amino acids have an HHPRED hit (98.8%)to DNA primase [PDB 5VAZ] amino acids 113 - 424 have HHPREd hit to INTEIN-ENCODED HOMING ENDONUCLEASE (99.9%) {PDB 1DQ3] and finally amino acids 414 - 513 also HHPRED (98.6%) the same DNA primase [PDB 5VAZ] most of the annotations in this pham are just DNA primase, one protein Turj99 gp53 is annotated (intein-containing topoisomerase primase). Clearly this is an intein within a primase, just not sure how best to annotate.

Link to this post | posted 18 Jun, 2018 02:55

cdshaffer

Not sure exactly how to annotate this gene from fushigi (37,851-36310 rev)
most A1 phage in this region have the two DNA primase genes with the large overlap, however in fushigi and a few others there is a single large gene of 513 amino acids:

The first 128 amino acids have an HHPRED hit (98.8%)to DNA primase [PDB 5VAZ]
amino acids 113 - 424 have HHPREd hit to INTEIN-ENCODED HOMING ENDONUCLEASE (99.9%) {PDB 1DQ3]
and finally amino acids 414 - 513 also HHPRED (98.6%) the same DNA primase [PDB 5VAZ]

most of the annotations in this pham are just DNA primase, one protein Turj99 gp53 is annotated (intein-containing topoisomerase primase).

Clearly this is an intein within a primase, just not sure how best to annotate.

Posted in: Functional Annotation → fushigi_draft 52 homing endonuclease + primase

Link to this post \| posted 16 Jun, 2018 22:53
cdshaffer	There are quite a few examples of two genes in a row being in the same pham. This is based on the decision (made very early on) as to what to do when you find a protein that aligns well to proteins in two different phams. The decision was made to combine the two phams together into a single larger pham and retire the two older phams. This usually happens by having one long protein where some smaller proteins align well to the first part of the long protein and other smaller proteins align to the second part of the larger protein. In the case of pham 24076 it looks like ShereKhan_Draft gene 75 is that larger protein. This situation is neither common or exceptionally rare. An example that has gotten a lot of discussion lately is the split lysin A proteins (do a forum search for "split lysin A" ) . This doesn't often help with functional annotation other than to explain why sometimes members of a pham can have two quite different functional annotations and both are justified by the evidence. Edited 16 Jun, 2018 22:53

Link to this post | posted 16 Jun, 2018 22:53

cdshaffer

There are quite a few examples of two genes in a row being in the same pham. This is based on the decision (made very early on) as to what to do when you find a protein that aligns well to proteins in two different phams. The decision was made to combine the two phams together into a single larger pham and retire the two older phams. This usually happens by having one long protein where some smaller proteins align well to the first part of the long protein and other smaller proteins align to the second part of the larger protein.

In the case of pham 24076 it looks like ShereKhan_Draft gene 75 is that larger protein. This situation is neither common or exceptionally rare. An example that has gotten a lot of discussion lately is the split lysin A proteins (do a forum search for "split lysin A" ) .

This doesn't often help with functional annotation other than to explain why sometimes members of a pham can have two quite different functional annotations and both are justified by the evidence.

Edited 16 Jun, 2018 22:53

Posted in: Functional Annotation → Two Pham 24076 genes in a row with different functions in cluster E

Link to this post \| posted 13 Jun, 2018 19:22
cdshaffer	Great questions. Pecaan does not save the actual alignments, it just saves the hit table with the results you see. If you want alignments you have to either use NCBI web based blast or DNA Master. As for the listing in the notes, the issue is the structure of the non-redundant database that is the default database everyone searches with BLAST. This database is made much smaller and easier to search by combining all identical sequences into a single entry. Thus if there are three phage genes with exactly the same amino acid sequence and you search the non-redundant protein database you will get one hit which will have three names. In the web NCBI blast results you will just see the title to one sequence and the rest are kind of hidden but you will see a link with a tiny triangle and word like "see 2 more Title(s)". If you click the link you would see the full descriptions for all the other proteins. For PECAAN it was decided to explicitly list out all three in the Description column since any one of the three genes could have the relevant functional annotation and thus all three are also used to create the full notes for the same reason. in your case the two submitted proteins "3-oxoadipate CoA-transferase" and "CoA transferase subunit A" are identical so they collapse into one entry in the non-redundant database with two descriptions. Edited 13 Jun, 2018 19:28

Link to this post | posted 13 Jun, 2018 19:22

cdshaffer

Great questions.

Pecaan does not save the actual alignments, it just saves the hit table with the results you see. If you want alignments you have to either use NCBI web based blast or DNA Master.

As for the listing in the notes, the issue is the structure of the non-redundant database that is the default database everyone searches with BLAST. This database is made much smaller and easier to search by combining all identical sequences into a single entry. Thus if there are three phage genes with exactly the same amino acid sequence and you search the non-redundant protein database you will get one hit which will have three names. In the web NCBI blast results you will just see the title to one sequence and the rest are kind of hidden but you will see a link with a tiny triangle and word like "see 2 more Title(s)". If you click the link you would see the full descriptions for all the other proteins.

For PECAAN it was decided to explicitly list out all three in the Description column since any one of the three genes could have the relevant functional annotation and thus all three are also used to create the full notes for the same reason.

in your case the two submitted proteins "3-oxoadipate CoA-transferase" and "CoA transferase subunit A" are identical so they collapse into one entry in the non-redundant database with two descriptions.

Edited 13 Jun, 2018 19:28

Posted in: PECAAN → Issues with BLAST results in PECAAN

Link to this post \| posted 05 Jun, 2018 19:51
cdshaffer	Many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but is rather a truncation. See this alignment as an example: Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence): `Query 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS Sbjct 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 Query 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK 112 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+ +FD +D+ P Sbjct 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG 120 Query 113 A 113 Sbjct 121 V 121` As per Cresawn et al PLoS ONE 10(3) these should be annotated MPME1 even though they are truncations.

Link to this post | posted 05 Jun, 2018 19:51

cdshaffer

Many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but is rather a truncation. See this alignment as an example:

Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence):

Query  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60
            MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS
Sbjct  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60

Query  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK  112
            TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+         +FD +D+ P
Sbjct  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG  120

Query  113  A  113

Sbjct  121  V  121

As per Cresawn et al PLoS ONE 10(3) these should be annotated MPME1 even though they are truncations.

Posted in: Cluster O Annotation Tips → MPME

Link to this post \| posted 05 Jun, 2018 14:46
cdshaffer	It appears many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but much more than a single residue difference: Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence): `Query 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS Sbjct 1 MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS 60 Query 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK 112 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+ +FD +D+ P Sbjct 61 TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG 120 Query 113 A 113 Sbjct 121 V 121` This is identical for 99 a.a. then what looks like a spurious BLAST extension, should we also call this MPME1 or add new approved term to somehow indicate a derivative of MPME1 As a follow up, in Cresawn et al PLoS ONE 10(3) they are annotating the "truncated" version with the full MPME1 annotation. I am going to add this to the O specific annotations forum Edited 05 Jun, 2018 19:45

Link to this post | posted 05 Jun, 2018 14:46

cdshaffer

It appears many of the O cluster phage have a derivative of MPME1, its clearly not a MPME2 but much more than a single residue difference:

Alignment to BPs_58 to JangDynasty gp22 (but several O phage have identical sequence):

Query  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60
            MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS
Sbjct  1    MFPITDTRREMTTMPTTEHGSDVQHLSPEHRDRAWRDRFNARWHYDYGGWIRTRPQDEAS  60

Query  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDML———————VVFD-YDTSPNK  112
            TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTD+         +FD +D+ P
Sbjct  61   TFALIPTKHYGPFTEDHSCPACLVVHPPEDCPVLSGNTDIFGTTSSGPTIFDSFDSGPGG  120

Query  113  A  113

Sbjct  121  V  121

This is identical for 99 a.a. then what looks like a spurious BLAST extension, should we also call this MPME1 or add new approved term to somehow indicate a derivative of MPME1
As a follow up, in Cresawn et al PLoS ONE 10(3) they are annotating the "truncated" version with the full MPME1 annotation. I am going to add this to the O specific annotations forum

Edited 05 Jun, 2018 19:45

Posted in: Cluster F Annotation Tips → MPMEs--which one?

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