SEA-PHAGES | All posts created by scaruso

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Link to this post \| posted 23 Nov, 2018 18:36
scaruso	Thank you. I see it in the text, now, though the table at the end shows "F." Thanks, again. Steve

Posted in: Functional Annotation → Portal/Head-t-o-tail Case Study Question

Link to this post \| posted 23 Nov, 2018 15:58
scaruso	There are no genes in the left (forward) part of the genome with transmembrane domains that haven't been assigned functions other than the one above. On the right side after all the forward genes: The first (stop = 24921) gene has a TMD according to PECAAN's TmHmm. It also has a poor HHPred hit to: PF16935.5 ; Hol_Tox ; Putative Holin-like Toxin (Hol-Tox) Probability: 25.57, E-value: 420.0, Score: 17.93, Aligned Cols: 34, Identities: 9%, Similarity: 0.036, PECAAN HHPred is a little different with a prob of 32.3. coverage is only 35.1 The next, oddball forward gene has four TM domains. It has been identified as a DNA binding protein and, curiously as Excise in another phage. I'm stuck on NKF without proof beyond those calls, though. So it could also be looked at. No HHPreds to anything like Holin, though, or anything else. The last has stop=39165, has 1 TMD, but is only 105 bp long. No significant HHPred hits on PECAAN or external. The only issue is, I used the TmHmm program in PECANN to screen all 84 genes. It caught the three above, but I don't know if it would catch every possibility. I don't know what others would be found with SOSUI. Edited 24 Nov, 2018 15:12

Link to this post | posted 23 Nov, 2018 15:58

scaruso

There are no genes in the left (forward) part of the genome with transmembrane domains that haven't been assigned functions other than the one above.

On the right side after all the forward genes:

The first (stop = 24921) gene has a TMD according to PECAAN's TmHmm. It also has a poor HHPred hit to:
PF16935.5 ; Hol_Tox ; Putative Holin-like Toxin (Hol-Tox)
Probability: 25.57, E-value: 420.0, Score: 17.93, Aligned Cols: 34, Identities: 9%, Similarity: 0.036,
PECAAN HHPred is a little different with a prob of 32.3. coverage is only 35.1

The next, oddball forward gene has four TM domains. It has been identified as a DNA binding protein and, curiously as Excise in another phage.
I'm stuck on NKF without proof beyond those calls, though. So it could also be looked at.
No HHPreds to anything like Holin, though, or anything else.

The last has stop=39165, has 1 TMD, but is only 105 bp long. No significant HHPred hits on PECAAN or external.

The only issue is, I used the TmHmm program in PECANN to screen all 84 genes.
It caught the three above, but I don't know if it would catch every possibility.
I don't know what others would be found with SOSUI.

Edited 24 Nov, 2018 15:12

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 22 Nov, 2018 18:01
scaruso	I'm following up on Zetzy, an A3, that we used in the Hack-a-thon, and I have a quick question. I was able to call two genes NKF (that had been labeled otherwise in other phages), and another as head-to-tail adapter based on the Case Study and the table provided at end, that outlines the PDB hit requirements (5A21_D). But the next gene's PDB his is to 5A21_E, which is not listed. The only 'E' version of the entry contains SPP1 proteins 15, 16, and 17. Any thoughts? 'G/17' is downstream, so could this be 'F/16"? HHPred: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13195 Protein: MSLLDGGPQYEDILVFPEEAVTDEDGNTKTRPSATGIPAKARFQVQGQSGTSARRAEQDNEGFESEKVYRMRFPRSWDAEHGVLGAQSEIEWRGKRWALFGDVNFYNSSRRTARIDYTVKRYZ

Link to this post | posted 22 Nov, 2018 18:01

scaruso

I'm following up on Zetzy, an A3, that we used in the Hack-a-thon, and I have a quick question.

I was able to call two genes NKF (that had been labeled otherwise in other phages), and another as head-to-tail adapter based on the Case Study and the table provided at end, that outlines the PDB hit requirements (5A21_D). But the next gene's PDB his is to 5A21_E, which is not listed. The only 'E' version of the entry contains SPP1 proteins 15, 16, and 17.

Any thoughts? 'G/17' is downstream, so could this be 'F/16"?

HHPred: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13195

Protein: MSLLDGGPQYEDILVFPEEAVTDEDGNTKTRPSATGIPAKARFQVQGQSGTSARRAEQDNEGFESEKVYRMRFPRSWDAEHGVLGAQSEIEWRGKRWALFGDVNFYNSSRRTARIDYTVKRYZ

Posted in: Functional Annotation → Portal/Head-t-o-tail Case Study Question

Link to this post \| posted 21 Nov, 2018 12:18
scaruso	Not in that area or its environs, there are a lot of identified genes on the left side, but I'll need to get back to you about the rest of the genome. The HHPred search was completed using only the normal DBs suggested by the manual. You might note, though, that it's common to get different results. I check all interesting ones by hand, still. See this example, where the PDB hit from HHPred on the web and on PECAAN had the same match, but very different probabilities (88.55 vs 55.6). https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13556 I'll be working on it over the next week and will update. Steve Edited 22 Nov, 2018 18:05

Link to this post | posted 21 Nov, 2018 12:18

scaruso

Not in that area or its environs, there are a lot of identified genes on the left side, but I'll need to get back to you about the rest of the genome.

The HHPred search was completed using only the normal DBs suggested by the manual. You might note, though, that it's common to get different results. I check all interesting ones by hand, still.

See this example, where the PDB hit from HHPred on the web and on PECAAN had the same match, but very different probabilities (88.55 vs 55.6). https://toolkit.tuebingen.mpg.de/#/jobs/Zet_13556

I'll be working on it over the next week and will update.

Steve

Edited 22 Nov, 2018 18:05

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 20 Nov, 2018 20:55
scaruso	I have what I believe is a decent candidate for the holin in the A3 subcluster, in Zetzy and would appreciate some feedback. It is immediately downstream of some tRNAs, and upstream of Lysin A and B. Such that, if correct, it would be: Terminase, small subunit Minor tail protein Minor tail protein NKF (probably a minor tail protein) tRNAs (x3) Holin Lysin A Lysin B Terminase, large subunit Portal Capsid maturation Scaffolding Major capsid protein The described synteny shows the Holin between Lysin A and Lysin B, but this seems like a reasonable variation, right in front of Lysin A and B. A link to the HHPred results can be found here: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_3980. I have attached a PDF of the result as well. SOSUI shows that it has a single transmembrane region near the N-terminus: LATIAKLIAQALLPIIAKQIAEE, and it is 255 aa long. The HHPred suggests it is a class VI holin, which has a single transmembrane region near the N-terminus, but it is also in line with the T4 (class III) family, which is closer in size. The gene product: MLATIAKLIAQALLPIIAKQIAEEFGKHVEPLTKALVTAVTEAAATGAERGADKLTDYIPGKLDDQIIDPIVKRGLEIFRDLTRZ Thanks! Steve Edited 21 Nov, 2018 12:15 3.44Mb

Link to this post | posted 20 Nov, 2018 20:55

scaruso

I have what I believe is a decent candidate for the holin in the A3 subcluster, in Zetzy and would appreciate some feedback.

It is immediately downstream of some tRNAs, and upstream of Lysin A and B. Such that, if correct, it would be:

Terminase, small subunit
Minor tail protein
Minor tail protein
NKF (probably a minor tail protein)
tRNAs (x3)
Holin
Lysin A
Lysin B
Terminase, large subunit
Portal
Capsid maturation
Scaffolding
Major capsid protein

The described synteny shows the Holin between Lysin A and Lysin B, but this seems like a reasonable variation, right in front of Lysin A and B.

A link to the HHPred results can be found here: https://toolkit.tuebingen.mpg.de/#/jobs/Zet_3980. I have attached a PDF of the result as well.

SOSUI shows that it has a single transmembrane region near the N-terminus: LATIAKLIAQALLPIIAKQIAEE, and it is 255 aa long. The HHPred suggests it is a class VI holin, which has a single transmembrane region near the N-terminus, but it is also in line with the T4 (class III) family, which is closer in size.

The gene product: MLATIAKLIAQALLPIIAKQIAEEFGKHVEPLTKALVTAVTEAAATGAERGADKLTDYIPGKLDDQIIDPIVKRGLEIFRDLTRZ

Thanks!

Steve

Edited 21 Nov, 2018 12:15

Posted in: Functional Annotation → A3 Holin?

Link to this post \| posted 20 Aug, 2018 17:22
scaruso	David Bollivar This is a tougher one. Again, just my two cents. The evidence points to this having a LysM domain, but that can be a little misleading because the LysM domain essentially is a peptidoglycan binding domain. Your CDD hit is just to that part of the predicted protein. Unfortunately, there is not an approved function with just the LysM domain, so the question is do you have enough evidence for the endolysin part. There are suggestions (the LD transpeptidase hit for example) but I would suggest it is not strong enough to make this call. I personally would leave this NKF. I can buy that. Thank you for your second look. Steve

Link to this post | posted 20 Aug, 2018 17:22

scaruso

David Bollivar
This is a tougher one. Again, just my two cents. The evidence points to this having a LysM domain, but that can be a little misleading because the LysM domain essentially is a peptidoglycan binding domain. Your CDD hit is just to that part of the predicted protein. Unfortunately, there is not an approved function with just the LysM domain, so the question is do you have enough evidence for the endolysin part. There are suggestions (the LD transpeptidase hit for example) but I would suggest it is not strong enough to make this call. I personally would leave this NKF.

I can buy that. Thank you for your second look.
Steve

Posted in: Functional Annotation → WheeHeim LysM

Link to this post \| posted 19 Aug, 2018 22:47
scaruso	Thank you. And thank you for the tip about GenemarkS on PDB, that's very useful. Steve

Posted in: Functional Annotation → WheeHeim Glycosyltransferase

Link to this post \| posted 19 Aug, 2018 22:45
scaruso	David Bollivar Steve, I have yet to look at the function aspect, but I would recommend you use the GenemarkS that you can run from phagesdb to look for coding potential. It has much more information to offer than what I see uploaded to PECAAN. Dave I'll check it out. Thank you. Steve

Posted in: Functional Annotation → WheeHeim Glycosyltransferase

Link to this post \| posted 19 Aug, 2018 22:27
scaruso	I am looking at WheeHeim_12322-12990, currently gp25 in PECAAN, and want to get a second opinion on a function call on it (an another gene in the previous post) As mentioned previously, WheeHeim is a singleton, and so is pretty weird looking. It has virtually no useful GM_Web coding potential, for example. Only three genes are in occupied phams, the rest are orphams. There are strong BlastP, HHPred, and CCD hits for lytic transglycoslyase and lytic murein transglycoslyase. That's certainly in line with an endolysin, and there are very. very poor hits to endolysin as well. The safest call would, of course be glycosyltransferase. But when I was searching in the HHPred results, one of the many lysozyme and other hits looked suggestive of lysin B. Could this be lysin B right after the Endolysin, LysM (lysin A) called two genes 5'? Any thoughts would be appreciated. 84Kb

Link to this post | posted 19 Aug, 2018 22:27

scaruso

I am looking at WheeHeim_12322-12990, currently gp25 in PECAAN, and want to get a second opinion on a function call on it (an another gene in the previous post)

As mentioned previously, WheeHeim is a singleton, and so is pretty weird looking. It has virtually no useful GM_Web coding potential, for example. Only three genes are in occupied phams, the rest are orphams.

There are strong BlastP, HHPred, and CCD hits for lytic transglycoslyase and lytic murein transglycoslyase. That's certainly in line with an endolysin, and there are very. very poor hits to endolysin as well. The safest call would, of course be glycosyltransferase.

But when I was searching in the HHPred results, one of the many lysozyme and other hits looked suggestive of lysin B. Could this be lysin B right after the Endolysin, LysM (lysin A) called two genes 5'?

Any thoughts would be appreciated.

Posted in: Functional Annotation → WheeHeim Glycosyltransferase

Link to this post \| posted 19 Aug, 2018 22:18
scaruso	I am looking at WheeHeim_11390-12103, currently gp23 in PECAAN, and want to get a second opinion on a function call on it (an another gene in the next post) WheeHeim is a singleton, and so is pretty weird looking. It has virtually no useful GM_Web coding potential, for example. Only three genes are in occupied phams, the rest are orphams. The BlastP and HHPred alignments for this gene are poor hits for 'endolysin, LysM-like,' but the CDD result is much stronger, something I rarely get to use. I think that makes the poor BlastP and HHPred hits more interesting and supportive. Any thoughts would be appreciated. Edited 19 Aug, 2018 22:50 88Kb

Link to this post | posted 19 Aug, 2018 22:18

scaruso

I am looking at WheeHeim_11390-12103, currently gp23 in PECAAN, and want to get a second opinion on a function call on it (an another gene in the next post)

WheeHeim is a singleton, and so is pretty weird looking. It has virtually no useful GM_Web coding potential, for example. Only three genes are in occupied phams, the rest are orphams.

The BlastP and HHPred alignments for this gene are poor hits for 'endolysin, LysM-like,' but the CDD result is much stronger, something I rarely get to use. I think that makes the poor BlastP and HHPred hits more interesting and supportive.

Any thoughts would be appreciated.

Edited 19 Aug, 2018 22:50

Posted in: Functional Annotation → WheeHeim LysM

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