SEA-PHAGES | All posts created by jparker

Link to this post \| posted 22 Dec, 2017 07:04
jparker	Paola (K5) may have a novel toxin/antitoxin system Gp86 (57,020-57,295) and Gp87 (57,297-57,725) – Structural models have led student team Sick Virions to believe that gp85 and gp86 belongs to a putative Toxin-Antitoxin system (TA system). Even though the two components resemble defined proteins from different TA systems, it has been suggested through structural analysis that the two different systems pose enough similarity between them so that the components are interchangeable while still being functional. Paola gp86 was reported as a type II BrnT toxin by the tool HHpred, which indicating that gp86 shares structural similarity with BrnT toxin from brucella abortus. Top two hits both call for this function with probability 99.7, good E-value and high coverage above 85%. 3D crystal structure of gp86 constructed by Phyre and Pymol suggests gp86 conserved the important beta-sheet domains as BrnT toxin has, which can interact with BrnA antitoxin. However, the sequence of gp86 does not align with that of BrnT toxin BlastX was used for the alignment. Gp86 has a length of 276bp, about the same length as BrnT toxin. In addition, gp87 shows structural similarity to RelB antitoxin, even though the coverage of this HHpred hit is low, Heaton et al (2012) indicates that BrnT has secondary structure highly resembles to RelE structure, the toxin paired with RelB, and BrnT and RelB may interact. This adds to the evidence of gp85 should be called for BrnT toxin. >Paola gp86 MARWADDRAEHLWERHEKTIEQAEEALNDPARVTLDPDPASKSGGGVRVVGYSPTAGCVLCVIVVPYEGELWGATAFPANKTYQRIYKEGAZ >Paola gp87 MSKKPNEELTRVLNNAALMAETDAEIMGDHVDLTDVKITRGGPRTRVLQIRLNDDELAELERQADDRDLPASTVAREILLRALFPRPAADPVAFSREGVAEALLRYVDAVVDRQLAARVEEVYRKGAEAFMVPGPAGYGSPMZ DNAM file for Paola is attached. Edited 22 Dec, 2017 07:04 32Kb

Link to this post | posted 22 Dec, 2017 07:04

Paola (K5) may have a novel toxin/antitoxin system

Gp86 (57,020-57,295) and Gp87 (57,297-57,725) –
Structural models have led student team Sick Virions to believe that gp85 and gp86 belongs to a putative Toxin-Antitoxin system (TA system). Even though the two components resemble defined proteins from different TA systems, it has been suggested through structural analysis that the two different systems pose enough similarity between them so that the components are interchangeable while still being functional.

Paola gp86 was reported as a type II BrnT toxin by the tool HHpred, which indicating that gp86 shares structural similarity with BrnT toxin from brucella abortus. Top two hits both call for this function with probability 99.7, good E-value and high coverage above 85%. 3D crystal structure of gp86 constructed by Phyre and Pymol suggests gp86 conserved the important beta-sheet domains as BrnT toxin has, which can interact with BrnA antitoxin. However, the sequence of gp86 does not align with that of BrnT toxin BlastX was used for the alignment. Gp86 has a length of 276bp, about the same length as BrnT toxin. In addition, gp87 shows structural similarity to RelB antitoxin, even though the coverage of this HHpred hit is low, Heaton et al (2012) indicates that BrnT has secondary structure highly resembles to RelE structure, the toxin paired with RelB, and BrnT and RelB may interact. This adds to the evidence of gp85 should be called for BrnT toxin.

>Paola gp86
MARWADDRAEHLWERHEKTIEQAEEALNDPARVTLDPDPASKSGGGVRVVGYSPTAGCVLCVIVVPYEGELWGATAFPANKTYQRIYKEGAZ

>Paola gp87
MSKKPNEELTRVLNNAALMAETDAEIMGDHVDLTDVKITRGGPRTRVLQIRLNDDELAELERQADDRDLPASTVAREILLRALFPRPAADPVAFSREGVAEALLRYVDAVVDRQLAARVEEVYRKGAEAFMVPGPAGYGSPMZ

DNAM file for Paola is attached.

Edited 22 Dec, 2017 07:04

Posted in: Request a new function on the SEA-PHAGES official list → Ribonuclease toxin BrnT

Link to this post \| posted 05 Sep, 2017 04:32
jparker	I think I have found a pham that is annotated incorrectly for the functions, and I wanted to post what I found, just in case it may help someone else. Pham 1929 (as of 9/4/17) in clusters L (Zakai_52) and O (Catdawg_81). In the cluster L, some of these are called membrane proteins or band-7 like, but I think they only have signal peptides, which can show up as transmembrane domains. Here is the TMHMM output for Zakai_52, but both Phobius and SignalP indicate signal peptides. For comparison, Catdawg_81, in the same pham, has similar Phobius and SignalP results, but no called transmembrane domains by TMHMM. HHPred did have high probability and high coverage hits to Hfl proteins, which is probably why membrane protein was called: High frequency of lysogenization C (HflC) family; SPFH (stomatin, prohibitin, flotillin, and HflK/C) superfamily This model characterizes proteins similar to prokaryotic HflC (High frequency of lysogenization C). Although many members of the SPFH (or band 7) superfamily are lipid raft associated, prokaryote plasma membranes lack cholesterol and are unlikely to have lipid raft domains. Individual proteins of this SPFH domain superfamily may cluster to form membrane microdomains which may in turn recruit multiprotein complexes. Escherichia coli HflC is an integral membrane protein which may localize to the plasma membrane. HflC associates with another SPFH superfamily member (HflK) to form an HflKC complex. HflKC interacts with FtsH in a large complex termed the FtsH holo-enzyme. FtsH is an AAA ATP-dependent protease which exerts progressive proteolysis against membrane-embedded and soluble substrate proteins. HflKC can modulate the activity of FtsH. HflKC plays a role in the decision between lysogenic and lytic cycle growth during lambda phage infection. Would it be helpful to have signal sequences noted in annotations, like we do for some domains? Edited 05 Sep, 2017 04:35

Link to this post | posted 05 Sep, 2017 04:32

jparker

I think I have found a pham that is annotated incorrectly for the functions, and I wanted to post what I found, just in case it may help someone else.

Pham 1929 (as of 9/4/17) in clusters L (Zakai_52) and O (Catdawg_81). In the cluster L, some of these are called membrane proteins or band-7 like, but I think they only have signal peptides, which can show up as transmembrane domains. Here is the TMHMM output for Zakai_52, but both Phobius and SignalP indicate signal peptides.

For comparison, Catdawg_81, in the same pham, has similar Phobius and SignalP results, but no called transmembrane domains by TMHMM. HHPred did have high probability and high coverage hits to Hfl proteins, which is probably why membrane protein was called:

High frequency of lysogenization C (HflC) family; SPFH (stomatin, prohibitin, flotillin, and HflK/C) superfamily
This model characterizes proteins similar to prokaryotic HflC (High frequency of lysogenization C). Although many members of the SPFH (or band 7) superfamily are lipid raft associated, prokaryote plasma membranes lack cholesterol and are unlikely to have lipid raft domains. Individual proteins of this SPFH domain superfamily may cluster to form membrane microdomains which may in turn recruit multiprotein complexes. Escherichia coli HflC is an integral membrane protein which may localize to the plasma membrane. HflC associates with another SPFH superfamily member (HflK) to form an HflKC complex. HflKC interacts with FtsH in a large complex termed the FtsH holo-enzyme. FtsH is an AAA ATP-dependent protease which exerts progressive proteolysis against membrane-embedded and soluble substrate proteins. HflKC can modulate the activity of FtsH. HflKC plays a role in the decision between lysogenic and lytic cycle growth during lambda phage infection.

Would it be helpful to have signal sequences noted in annotations, like we do for some domains?

Edited 05 Sep, 2017 04:35

Posted in: Functional Annotation → Signal peptides vs membrane proteins

Link to this post \| posted 25 Aug, 2017 23:20
jparker	Hello, The Cluster C1 phages I have been annotating are circularly permuted and have a gene that appears to wrap around the end of the genome. Is there anything else that needs to be done when annotating these? I'm assuming it should be changed to "CDS" from "misc_signal"? This is what it looks like in the documentation: misc_signal join(155400. .155759;1. .1 /gene="259" /product="gp259" /locus tag="KOGUMA_259" Thanks! Jordan

Link to this post | posted 25 Aug, 2017 23:20

jparker

Hello,
The Cluster C1 phages I have been annotating are circularly permuted and have a gene that appears to wrap around the end of the genome. Is there anything else that needs to be done when annotating these? I'm assuming it should be changed to "CDS" from "misc_signal"?

This is what it looks like in the documentation:
misc_signal join(155400. .155759;1. .1 smile

/gene="259"
/product="gp259"
/locus tag="KOGUMA_259"

Thanks!
Jordan

Posted in: Cluster C Annotation Tips → wrap around genes in C1

Link to this post \| posted 25 Aug, 2017 04:22
jparker	The Cluster C1 phages have strong support HHPred for thymidylate kinase, and some people have already been calling it (but it is not on the list). See Daffodil_202 or Erdmann_199.

Posted in: Request a new function on the SEA-PHAGES official list → thymidylate kinase

Link to this post \| posted 21 Aug, 2017 22:53
jparker	Hi Chris, The pham links on PECAAN to Starterator and phagesDB phams don't link to the same actual groups of genes. Web Phamerator seems to match to the phagesDB pham, so I can't find the new corresponding pham numbers in starterator. The starterator build date is 8/16/17, but I'm not sure which one the web phamerator and PhagesDB phams are on. Any suggestions to connecting these back together? Thanks! Jordan

Link to this post | posted 21 Aug, 2017 22:53

jparker

Hi Chris,
The pham links on PECAAN to Starterator and phagesDB phams don't link to the same actual groups of genes. Web Phamerator seems to match to the phagesDB pham, so I can't find the new corresponding pham numbers in starterator. The starterator build date is 8/16/17, but I'm not sure which one the web phamerator and PhagesDB phams are on. Any suggestions to connecting these back together?

Thanks!
Jordan

Posted in: Starterator → Updates to Starterator output

Link to this post \| posted 18 Aug, 2017 01:42
jparker	Hi All, If we think we have identified the attP site, should we annotate it? And if so, how? For example, for Sulley (K1): AttP Site Start: 32614 Stop: 32641 Sequence: ggttcaattcccggcagctccacgagta Thanks! Jordan

Posted in: Annotation → annotating attP site

Link to this post \| posted 17 Aug, 2017 01:44
jparker	A bunch of genes in pham 609, such as Eremos_49 and other Cluster B1 phages are labeled as Lysin B. HHPred seems to support this with hits to Cutinase. However, what I cannot find is Holin! The genes called as lysin B do have 3 transmembrane domains, so I am very confused!

Posted in: Cluster B Annotation Tips → Lysin B

Link to this post \| posted 02 Aug, 2017 00:28
jparker	Hello, Last year Welkin mentioned that it is possible to export or import stuff from the DNAM "Submit Sequence to Genbank" tool. She mentioned this in the context of having students do the tedious data entry bits, which is exactly what I want to do. Can someone point me to the instructions to do this? Thanks! Jordan

Posted in: Notes and Final Files → Exporting DNAM "Submit to Genbank" stuff

Link to this post \| posted 01 Aug, 2017 03:05
jparker	Just a thought, but I usually think of these as transmembrane "domains" rather than proteins. What is the cutoff for labeling something a particular kind of protein, compared to a protein with a particular domain? I attached a screenshot of the TMHMM output for Emma_35 (Pham 12413 - which appears to be conserved in F1 and BD clusters). So, we are going with "predicted membrane protein"? 57Kb

Posted in: Request a new function on the SEA-PHAGES official list → membrane protein

Link to this post \| posted 26 May, 2017 02:13
jparker	I have tried, but the new HHPred site refuses to work for me. Connection errors, freezing, just refusing to have anything happen which I click buttons. Has anyone gotten it to work? Jordan

Posted in: Annotation → New HHPred web site

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