SEA-PHAGES | All posts created by fbaliraine

Link to this post \| posted 15 Jul, 2022 21:44
fbaliraine	Phage Skinny gene at 63560-63949 bp hits several LAGLIDADG endonuclease, homing endonuclease, HNH endonuclease, with low similarity (less than 60%). Wondering whether to call it with the general “Hydrolase” or “NKF”? The amino acid sequence is: MDLAYLGGFFDGEGNVGLYKSGGESPRLRVQVFQNHGASQDRLMHEIHDTFGGTLHDRGTGYLYSASGSRAVDLLTQLRPHLRLKLEQADEALEWWRNRTAERFRSRTAEEVAYDESAMTRLKELKRAGZ I have also attached the relevant BLASTp and HHPred data: Thanks! 460Kb

Link to this post | posted 15 Jul, 2022 21:44

Phage Skinny gene at 63560-63949 bp hits several LAGLIDADG endonuclease, homing endonuclease, HNH endonuclease, with low similarity (less than 60%). Wondering whether to call it with the general “Hydrolase” or “NKF”? The amino acid sequence is:
MDLAYLGGFFDGEGNVGLYKSGGESPRLRVQVFQNHGASQDRLMHEIHDTFGGTLHDRGTGYLYSASGSRAVDLLTQLRPHLRLKLEQADEALEWWRNRTAERFRSRTAEEVAYDESAMTRLKELKRAGZ
I have also attached the relevant BLASTp and HHPred data:
Thanks!

Posted in: Functional Annotation → “Hydrolase” or “NKF” for hits to LAGLIDADG endonuclease, homing endonuclease, HNH endonuclease?

Link to this post \| posted 09 May, 2022 19:05
fbaliraine	Thanks. I’ve looked at the “Thioredoxin” link that you’ve kindly provided, but there is something noteworthy in HHPred. In PDB, the reference sequence phage Onyinye gene78 for “oxidoreductase” is almost thrice as long 792 bp vs 273 bp of Gilberta) and mostly hits “Polyketide oxygenase PgaE,” chain A, with ref Sequence in pfam hitting the "FAD folding domain" but I do not see any hits in Gilberta to any "FAD folding domain." Instead, in PDB Gilberta hits the "Molecule" THIOREDOXIN chains A & B, with the pfam ref hitting “Glutaredoxin or Glutaredoxin-like NRDH-redoxin, THIOREDOXIN; OXIDOREDUCTASE, GLUTAREDOXIN.” On the other hand, the ref sequence for Thioredoxin, phage Cjw1 gp 37 (246 bp) has a comparable length to Gilberta’s 273 bp and they both have several hits the molecule Thioredoxin chain A, but not the “Polyketide oxygenase PgaE,” or “FAD domain”. Edited 09 May, 2022 19:08

Link to this post | posted 09 May, 2022 19:05

fbaliraine

Thanks. I’ve looked at the “Thioredoxin” link that you’ve kindly provided, but there is something noteworthy in HHPred.

In PDB, the reference sequence phage Onyinye gene78 for “oxidoreductase” is almost thrice as long 792 bp vs 273 bp of Gilberta) and mostly hits “Polyketide oxygenase PgaE,” chain A, with ref Sequence in pfam hitting the "FAD folding domain" but I do not see any hits in Gilberta to any "FAD folding domain." Instead, in PDB Gilberta hits the "Molecule" THIOREDOXIN chains A & B, with the pfam ref hitting “Glutaredoxin or Glutaredoxin-like NRDH-redoxin, THIOREDOXIN; OXIDOREDUCTASE, GLUTAREDOXIN.” On the other hand, the ref sequence for Thioredoxin, phage Cjw1 gp 37 (246 bp) has a comparable length to Gilberta’s 273 bp and they both have several hits the molecule Thioredoxin chain A, but not the “Polyketide oxygenase PgaE,” or “FAD domain”.

Edited 09 May, 2022 19:08

Posted in: Functional Annotation → Function for subcluster A11 phage Gilberta (37505-37777 rev): Thioredoxin, NrdH-like glutaredoxin or glutaredoxin?

Link to this post \| posted 08 May, 2022 02:08
fbaliraine	There is a "mixed bag" with many hits to NrdH-like glutaredoxin and glutaredoxin in phagesDB, with a few hits to Thioredoxin for this Gilberta sequence MRTMFAPITIYTQPRCAPCDALKKRLEKEGIAFDAVDITKNEEAYAYVTGVLKAAATPIIVTDTHDPIIGDRPAELEELIEYYTTSETGVZ However, the PDB HHPred shows more than 22 hits to Thioredoxin with alignments of 78-93% and probability of 99%, versus less than 5 hits to NrdH-like glutaredoxin with alignments ranging from 81-85% and probability of 99%, besides hits to glutaredoxin, all three of which are options currently provided in the Official Functions list. Should we go for, “Thioredoxin” or use “NrdH-like glutaredoxin” or the general term "glutaredoxin"? In the “Cluster EB/ED glutaredoxin” forum post of 06 May, 2019, the use of “NrdH-like glutaredoxin” was considered appropriate for “HHPRED hit with >98% prob and >98% coverage to 4FIW_A which is the published crystal structure of NrdH from E coli.” See details of hits below: HHPred PDB hits to Thioredoxin (6MOS_A, 93.4% alignment, 99.14% probability; 7ASW_A, 91.2% alignment, 98.91% probability; 3ZIT_B, 84.62% alignment, 99.47% probability; d1nhoa_, 85.71 alighment, 99.23% probability; d1f9ma_, d1ti3a_, d1r26a1, & d1ep7a_, 86.81 alignment, 99.2% probability; d4oo4a_, 85.71 alignment, 99.18% probability; d4j56e1 & d1thxa_, 85.71 % alignment, 99.13% probability; 3KP8_A, 84.61% alignment, 99.12% probability; d1nw2a_, 84.61% alignment, 99.02% probability; d1iloa_ c, 78.02% alignment, 99.1% probability; d3diea1, 83.52% alignment, 99.1% probability; 7B02_A, 86.81% alignment, 99.1% probability; 3HZ4_A & 7RGV_A, 90.1% alignment, 99.1% probability; 7BZK_B, 6ZYW_P, 6I1C_B, & 6Q6T_A, 89.01% alignment, 99.0% probability; 1THX_A, 87.91% alignment, 99.02% probability). HHPred hits to NrdH-like glutaredoxin in (d1r7ha_, 81.32% alignment, 99.49% probability; d1h75a_, 83.52% alignment, 99.46 probability; 1R7H_A, 81.32% alignment, 99.11% probability; 4K8M_A, 84.61% alignment, 98.99% probability). Thanks! Edited 08 May, 2022 02:16

Link to this post | posted 08 May, 2022 02:08

fbaliraine

There is a "mixed bag" with many hits to NrdH-like glutaredoxin and glutaredoxin in phagesDB, with a few hits to Thioredoxin for this Gilberta sequence MRTMFAPITIYTQPRCAPCDALKKRLEKEGIAFDAVDITKNEEAYAYVTGVLKAAATPIIVTDTHDPIIGDRPAELEELIEYYTTSETGVZ
However, the PDB HHPred shows more than 22 hits to Thioredoxin with alignments of 78-93% and probability of 99%, versus less than 5 hits to NrdH-like glutaredoxin with alignments ranging from 81-85% and probability of 99%, besides hits to glutaredoxin, all three of which are options currently provided in the Official Functions list. Should we go for, “Thioredoxin” or use “NrdH-like glutaredoxin” or the general term "glutaredoxin"?

In the “Cluster EB/ED glutaredoxin” forum post of 06 May, 2019, the use of “NrdH-like glutaredoxin” was considered appropriate for “HHPRED hit with >98% prob and >98% coverage to 4FIW_A which is the published crystal structure of NrdH from E coli.” See details of hits below:
HHPred PDB hits to Thioredoxin (6MOS_A, 93.4% alignment, 99.14% probability; 7ASW_A, 91.2% alignment, 98.91% probability; 3ZIT_B, 84.62% alignment, 99.47% probability; d1nhoa_, 85.71 alighment, 99.23% probability; d1f9ma_, d1ti3a_, d1r26a1, & d1ep7a_, 86.81 alignment, 99.2% probability; d4oo4a_, 85.71 alignment, 99.18% probability; d4j56e1 & d1thxa_, 85.71 % alignment, 99.13% probability; 3KP8_A, 84.61% alignment, 99.12% probability; d1nw2a_, 84.61% alignment, 99.02% probability; d1iloa_ c, 78.02% alignment, 99.1% probability; d3diea1, 83.52% alignment, 99.1% probability; 7B02_A, 86.81% alignment, 99.1% probability; 3HZ4_A & 7RGV_A, 90.1% alignment, 99.1% probability; 7BZK_B, 6ZYW_P, 6I1C_B, & 6Q6T_A, 89.01% alignment, 99.0% probability; 1THX_A, 87.91% alignment, 99.02% probability).

HHPred hits to NrdH-like glutaredoxin in (d1r7ha_, 81.32% alignment, 99.49% probability; d1h75a_, 83.52% alignment, 99.46 probability; 1R7H_A, 81.32% alignment, 99.11% probability; 4K8M_A, 84.61% alignment, 98.99% probability).
Thanks!

Edited 08 May, 2022 02:16

Posted in: Functional Annotation → Function for subcluster A11 phage Gilberta (37505-37777 rev): Thioredoxin, NrdH-like glutaredoxin or glutaredoxin?

Link to this post \| posted 06 May, 2022 18:27
fbaliraine	Thank you Debbie! Fred

Posted in: Functional Annotation → A small minor tail protein called based on solely on synteny?

Link to this post \| posted 06 May, 2022 17:34
fbaliraine	In the subcluster A11 phage Gilberta, we are seeing a small (189 bp long; position 25381-25569) gene hitting more than 60 minor tail protein genes in both NCBI and phagesDB. This gene is right downstream of a large (1992 bp long) minor tail protein gene which follows other minor tail proteins upstream of it. However, this small (189 bp) gene has neither hits to collagen-like, glycine-rich proteins, coiled-coils, nor any other significant hits in HHPred (Only one HHPred hit to Bacteriophage FRD2 protein, with 41.2% alignment and 11.9% probability). Could we still call it a minor tail protein solely based on synteny? Below is its amino acid sequence: MPWSPSPAFPQRQHRTAWFAELPAPTPAQHQTAWWAVYELDAPVEIACVTAAEGQEGPEEAVZ Thanks! Fred Edited 06 May, 2022 19:16

Link to this post | posted 06 May, 2022 17:34

fbaliraine

In the subcluster A11 phage Gilberta, we are seeing a small (189 bp long; position 25381-25569) gene hitting more than 60 minor tail protein genes in both NCBI and phagesDB. This gene is right downstream of a large (1992 bp long) minor tail protein gene which follows other minor tail proteins upstream of it. However, this small (189 bp) gene has neither hits to collagen-like, glycine-rich proteins, coiled-coils, nor any other significant hits in HHPred (Only one HHPred hit to Bacteriophage FRD2 protein, with 41.2% alignment and 11.9% probability). Could we still call it a minor tail protein solely based on synteny? Below is its amino acid sequence: MPWSPSPAFPQRQHRTAWFAELPAPTPAQHQTAWWAVYELDAPVEIACVTAAEGQEGPEEAVZ
Thanks!
Fred

Edited 06 May, 2022 19:16

Posted in: Functional Annotation → A small minor tail protein called based on solely on synteny?

Link to this post \| posted 21 Apr, 2022 23:19
fbaliraine	Hi Debbie, I concur. Case closed! Thank you for critically looking at this and clearing the air about calling genes that overlap with tRNAs. Perhaps a note in the resource guide can help eliminate future possibilities of someone calling such protein genes simply based on previous calls or significant BLASTp matches. Fred

Posted in: tRNAs → Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?

Link to this post \| posted 19 Apr, 2022 02:30
fbaliraine	Hi Debbie, Please find the DNA Master file attached. The tRNAs look legit, but the question is whether to delete the reverse gene at 92938-93090 bp, given that it is currently in pham 14023 with 31 members and hits various phage genes (including very recently annotated phages such as Ronan gp 160, GenBank submission 01-JUN-2020, and Mangeria GenBank Accn # YP_010057792.1) in phagesDb with q1:s1, 100% identity and e-values such as 6e-22. Thanks, Fred 811Kb

Link to this post | posted 19 Apr, 2022 02:30

fbaliraine

Hi Debbie,
Please find the DNA Master file attached. The tRNAs look legit, but the question is whether to delete the reverse gene at 92938-93090 bp, given that it is currently in pham 14023 with 31 members and hits various phage genes (including very recently annotated phages such as Ronan gp 160, GenBank submission 01-JUN-2020, and Mangeria GenBank Accn # YP_010057792.1) in phagesDb with q1:s1, 100% identity and e-values such as 6e-22.
Thanks,
Fred

Posted in: tRNAs → Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?

Link to this post \| posted 14 Apr, 2022 21:52
fbaliraine	I am asking this question to have this issue settled and possibly suggest that a note be included in the Resource Guide regarding tRNA annotation. Phage IkeLoa reverse draft gene 134 at 92938-9390 bp overlaps a tRNA by 27 bp; even if we change the start, it still overlaps by a few bp. Its sequence is: MLYQLSYDGGAAGETSQPSRLRVMSPVCLPSTTIPARVTDEDLTFVKPLSZ According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.” We have been following this rule and I am inclined to delete this protein gene, but I want to be sure in case something has changed recently, because it is matching q1:s1, 100% with recently annotated genes in phagesDB and phamerator (years 2017-2020; see attached). Thanks! Fred 267Kb

Link to this post | posted 14 Apr, 2022 21:52

fbaliraine

I am asking this question to have this issue settled and possibly suggest that a note be included in the Resource Guide regarding tRNA annotation.

Phage IkeLoa reverse draft gene 134 at 92938-9390 bp overlaps a tRNA by 27 bp; even if we change the start, it still overlaps by a few bp. Its sequence is: MLYQLSYDGGAAGETSQPSRLRVMSPVCLPSTTIPARVTDEDLTFVKPLSZ

According to the forum post, “How close can one pack protein and tRNA's genes” of Feb 24, 2016, Dr Pope stated that, “We tend to steer clear of a tRNA and a protein occupying the same space, but there are definitely genomes where they get pretty close.”
We have been following this rule and I am inclined to delete this protein gene, but I want to be sure in case something has changed recently, because it is matching q1:s1, 100% with recently annotated genes in phagesDB and phamerator (years 2017-2020; see attached).
Thanks!
Fred

Posted in: tRNAs → Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?

Link to this post \| posted 15 Feb, 2022 21:45
fbaliraine	chg60 Hi Debbie and Fred, I've attempted to begin a systematic analysis to determine how much we can trust the outputs from either of these programs. I accumulated a list of diverse types of DNA binding proteins: tyrosine or serine integrases, terminase large subunits, HTH DNA-binding proteins, RecE exonuclease, RecT ssDNA binding protein, etc. I pulled representative sequences from a subset of phams predominated by proteins with these functions. With the caveat that I've only run 6 sequences so far, I'm not impressed by DNABIND. It's very fast (which is nice!), but only two of the sequences were predicted as DNA-binding proteins (a tyrosine integrase and an HTH DNA-binding protein). The others were all reported as having a probability less than 40% of being DNA-binding. DNABINDER is MUCH slower - I'm still waiting on the first protein sequence, nearly an hour later. Ignoring the question of whether we can trust its output, I'm of the opinion that this program is too slow to warrant systematic use by SEA-PHAGES annotators. -Christian Hi Christian, DNABINDER will eat your lunch if you leave the setting as PSSM! If you want to go home earlier, please change the selection to "amino acid composition." The PSSM model is looking at the evolutionary trends, which is why it takes so long; amino acid composition is predicting on the basis of the amino acid composition like we are wanting (more details are explained on their website). Cheers! Fred

Link to this post | posted 15 Feb, 2022 21:45

fbaliraine

chg60
Hi Debbie and Fred,

I've attempted to begin a systematic analysis to determine how much we can trust the outputs from either of these programs.

I accumulated a list of diverse types of DNA binding proteins: tyrosine or serine integrases, terminase large subunits, HTH DNA-binding proteins, RecE exonuclease, RecT ssDNA binding protein, etc. I pulled representative sequences from a subset of phams predominated by proteins with these functions.

With the caveat that I've only run 6 sequences so far, I'm not impressed by DNABIND. It's very fast (which is nice!), but only two of the sequences were predicted as DNA-binding proteins (a tyrosine integrase and an HTH DNA-binding protein). The others were all reported as having a probability less than 40% of being DNA-binding.

DNABINDER is MUCH slower - I'm still waiting on the first protein sequence, nearly an hour later. Ignoring the question of whether we can trust its output, I'm of the opinion that this program is too slow to warrant systematic use by SEA-PHAGES annotators.

-Christian

Hi Christian,
DNABINDER will eat your lunch if you leave the setting as PSSM! If you want to go home earlier, please change the selection to "amino acid composition." The PSSM model is looking at the evolutionary trends, which is why it takes so long; amino acid composition is predicting on the basis of the amino acid composition like we are wanting (more details are explained on their website).
Cheers!
Fred

Posted in: Functional Annotation → Can we call DNA Binding proteins based on DNABIND and DNA Binder results?

Link to this post \| posted 15 Feb, 2022 21:38
fbaliraine	debbie Hi Fred, I don't know enough about DNABIND or DNA Binder to know how good that they are predicting DNA binding proteins. An analysis of what we have called DNA binding proteins with these programs is in order to determine if we would want to adopt this, I think. Make sense? debbie Hi Debbie, I concur with you that "An analysis of what we have called DNA binding proteins with these programs is in order to determine if we would want to adopt this." For now, I would settle for NKF until we get more support. Christian's current work is in order, and we too are starting on preliminary work to confirm. Fred

Link to this post | posted 15 Feb, 2022 21:38

fbaliraine

debbie
Hi Fred,
I don't know enough about DNABIND or DNA Binder to know how good that they are predicting DNA binding proteins. An analysis of what we have called DNA binding proteins with these programs is in order to determine if we would want to adopt this, I think.
Make sense?
debbie

Hi Debbie,
I concur with you that "An analysis of what we have called DNA binding proteins with these programs is in order to determine if we would want to adopt this."
For now, I would settle for NKF until we get more support. Christian's current work is in order, and we too are starting on preliminary work to confirm.
Fred

Posted in: Functional Annotation → Can we call DNA Binding proteins based on DNABIND and DNA Binder results?

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