Link to this post | posted 30 May, 2023 17:39
fbaliraine	Thank you Debbie. This clarifies things about this gene since it has previously been called NKF as evident from the phagesDB hits. We are now calling this large gene (gp 35 in Dulcita and Diminimus and other homologous subclcuster M1 phage genes in the same region) as minor tail protein, and the small gene (gp 30) as NKF. Fred Edited 30 May, 2023 17:40

Link to this post | posted 30 May, 2023 05:45

fbaliraine

We are annotating this large protein (999 aa) down the tape measure, in is draft gp 35 in both phages Dulcita and Diminimus (28791-29789 bp) in subcluster M1. It’s sequence is below: MASFTPIVPVRSNRPLELQRGNTLRYRYTLSGNKTFPAGTSAVLTVSNTYGQVVGAFIGTVAGKTIEFVEGPEISDTLARTDTWTLSVTYPGETHPTMLEQGQIIRVEAPFPDQPAMSPEFEGVRYEYHFGTPGFVKDPSWRILNGHPRVYDNSFRSLPNAVSSGSLFGGDLTFFDDVCMLWFAPLATDIVRLTYNTIRPIDNSNGEVWTIICSNYDATNWAGFHHKQVFGIGSWDDDEISVVTGTGPTTFTKRETESYDTVNNQAYTAEYNPVSNTYSLYVGTSLEPLISWTDETNVVEHGEGERYVGFGFKSALLYAGVQVSDWYIANTPZ Notably, it mostly hits hypothetical proteins in phagesDB, although it hits minor tail proteins of the same phages such as PegLeg & Reindeer in NCBI (which happen to show hypothetical proteins in phagesDB). In HHPred, it hits gene 31 of Mycobacterium phage D29 and phage L5, which upon inspection in the phagesDB gene list, that gene is a minor tail protein, and the HH probabilities for both phages are 99.85% & 99.86% respectively, with the hit in UniProt note about Protein existence, being "Predicted" (In some case for other proteins, UniProt states, "Evidence at protein level". According to the Resource Guide, minor tail proteins genes are big genes down the tape measure and usually not more than 5. We think that this would make the 5th big gene after the tape measure. We also know that we can use synteny only to call minor tail proteins, “You can call minor tail proteins for the 'big' genes downstream of the tape measure protein. there is usually not more than 5.” To use synteny, ALL the following three conditions must be met: (1) of the correct size , (2) adjacent to other structural genes of known, verifiable function and (3) the only possible option for that function in the genome (https://seaphagesbioinformatics.helpdocsonline.com/article-90). Given that D29 is a well-studied prototypic phage, we are inclined to call this large gene a minor tail protein, but since many hits in phagesDB are to Hypothetical proteins, we would like to cross-check to be sure that we are not missing something. On the other hand, there is a smaller (459 aa) draft gene 30 in Diminimus and Ducita downstream the tape measure which hits many minor tail proteins in phagesDB and is between two large, minor tail proteins but we would contend that this should be a Hypothetical Protein given its small size. Its sequence is: MPPLNVHPPDPNHPKGMAWVLGVGMVDPRPGNNPNQPMAIVQSWEPTSELWWKLGLRWHPELAEVWAVGGGQFEIAQIVNEKPEAQEMSLEEGAAEVLEYIGKEHPEYAEMLQQIHNAGSDVERIKLVKQFDGEIKRLMTLMKYVSTKPAEEZ Except something has changed lately, the following forum posts (4464 & 4546) suggest not calling small proteins minor tail proteins (https://seaphages.org/forums/topic/4464/; https://seaphages.org/forums/topic/4546/) In summary, we are thinking of calling the large (999 aa) gene (draft gp 35) a minor tail protein and the smaller (459 aa) gene (draft gp 30) a hypothetical protein. See attached phamerator map. What’s your verdict? Fred Edited 30 May, 2023 17:41 115Kb

Link to this post | posted 13 May, 2023 00:14
fbaliraine	Thank you Debbie & Chris! I think a note such as “Has H-N-H within 30-40 aa span but minor variations such as HNK, HNN, HNNH allowed, see forum topic 5505” or something similar as Chris suggests would be very helpful. Fred

Link to this post | posted 09 May, 2023 19:10

fbaliraine

Thank you Debbie & Christopher. I think the simple test would be very helpful, as the PDB hits were matching the Reference sequences as illustrated in the attachment with my initial post. I have edited the initial post to clarify that I was not referring to NCBI Ref sequences, but rather to the reference sequences provided in the Official Function List. The edit is, "The reference sequences for HNH endonuclease provided in the Official SEA-PHAGES Function List (as of May 9, 2023)." If SMART could clarify the "HNNH" pointed out by Christopher, that would be great as well. I have also taken another look at Glaske16 gp 98 at position 56773-57150 bp which Christopher pointed out that it is not an HNH endonuclease(see attached) and note that it has HNNH in a 35 aa span (not exactly HNH), whereas the Official Function List states that HNH endonuclease “Must have H-N-H over a 30 aa span.” If we henceforward won’t be calling this an HNH endonuclease, since many previous annotators have called it HNH (Glaske16 gp98 has more than 60 hits to HNH endonuclease in phagesDB!), could it help to state in the Official Function List that for any gene to be called an HNH endonuclease, it “Must have H-N-H within a span of not more than 30 aa,” besides clarifying whether H-N-N-H could also be acceptable if it is within a 30 aa span? Thanks! Fred Edited 10 May, 2023 05:01 586Kb

Link to this post | posted 04 May, 2023 23:30

fbaliraine

This is a loaded multi-question but please bear with me! HNH is expected to have a typical ββα-metal fold and Zn-finger motif (which would need protein modeling software to decipher; DOI: https://doi.org/10.1038/srep42542), and the Official Function List simply states that it “Must have H-N-H over a 30 aa span.” It would help students if there was an easy way to make a determination on this since it may not always be obvious in HHPred. Besides just considering the percent probability, should we also consider the e-values (and probably have an e-value cut-off)? Additionally, must it always hit chain A as well as the Zn-finger motif, or could it hit other chains such as chain D, with non-zinc motifs such as for Manganese or strontium ions? In view phagesDB & HHPred data, we are seeking clarification of the HNH function status of the following five draft Glaske16 genes at positions: 44853-45341 bp (gp 70), 51656-52198 bp (gp 83), 54100-54426 bp (gp 91), 56773-57150 bp (gp 9, and 60940-61320 bp (gp 117). Their respective sequences are provided, along with background information. >Glaske16_gp70_(44853-45341 bp) MPDGNQPACKYGACNDPVLARGFCKLHYYRNRDGKPMDGPRRSYSTGPRAWTYERLASVPITSTGAHQRVRRLWGSASLYPCATCGGPAKDWAYDGTDPTHYYEQGRKAWSHFSRWPEFYMPMCKPCHSNHDRRAAADELREYRQWKMRNPGKTLEDLEGVAZ >Glaske16_gp83_(51656-52198 bp) MDTIWKPIPQDPTGLYLASQDGRILRKEYVIEKLQSHGHLYRRVMPEKIVKQCIKDRAPSHGVHPIIQMRSSTQYASTVERRVSSLIAAAWHGLPYEAGDRTAQNDWRIGFIDGDPSNVHADNLEWVSNQGVNTHHSHDFYYENLKAYRAQAAVETAESFLARYYSPDEIDWSTAERIAAZ >Glaske16_gp91_( 54100-54426 bp) MPTNSKNGPRSRGRTGGKFERAKWRVLKANQICAHPDCRQLIDLDLKWPDPMSPTVNHIIPVKDLAWDDPLTYSVENLEPMHLVCNQRLGAGPRKKKPKHPQSRNWREZ >Glaske16_gp98_(56773-57150 bp) MALAGEAKREYQRQWRANRRAAWFAGKACVRCGSDEDLELDHVDPTLKVTNAVWSWSQERRDVELAKCQVLCNACHKAKTISQTVITIGLKAYRHGTCSMYEHHRCRCGLCRLWARNKKRRQRAAZ >Glaske16_gp117_(60940-61320 bp) MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVEZ The reference sequences for HNH endonuclease provided in the Official SEA-PHAGES Function List (as of May 9, 2023) are Sisi gp 99 and Arianna gp 54. Both match Geobacillus virus E2 hit 5H0M_A in PDB, with Sisi having a 93.5% alignment, 98.7% probability, and E-value: 1e-7, while Arianna has 67.3% alignment, 98.7% probability, and E-value: 2.2e-7. >Sisi_gp99 MPRAPKVCRHAGCTTLTTTGTCPQHTTHRWGNHQGRKVPHRLQQATFRRDNWTCQSCGHTATPGSGQLHADHIQPRSRGGADTLDNMRTLCKACHAPKSRAEARGSNT > Arianna_gp54 MAWSNGSSRTSSKHWQALRASAKKQLGYYCCAVCGITPAGGARLELDHIIPVAEGGSDEMANLQWLCARHHAIKTRAESRRGAQRRAARRRLPQRPHPGLR HHPred for Arianna is: PDB, Geobacillus virus E2, hit # 5H0M_A, 67.3% alignment, Probability: 98.67%, E-value: 2.2e-7. In view of the above, we can now specifically ask about the following five draft Glaske16 genes at positions: 44853-45341 bp (gp 70), 51656-52198 bp (gp 83), 54100-54426 bp (gp 91), 56773-57150 bp (gp 9, and 60940-61320 bp (gp 117). Glaske16 gp 70 (44853-45341 bp has the top PhagesDb hit as Skinny gp 71 which is called Hypothetical Protein, yet it is 100% identical, q1:s1, but has >10 hits to HNH endonuclease). I am inclined to call this an HNH endonuclease, except if the forum suggests otherwise. Again, below is its aa sequence: MPDGNQPACKYGACNDPVLARGFCKLHYYRNRDGKPMDGPRRSYSTGPRAWTYERLASVPITSTGAHQRVRRLWGSASLYPCATCGGPAKDWAYDGTDPTHYYEQGRKAWSHFSRWPEFYMPMCKPCHSNHDRRAAADELREYRQWKMRNPGKTLEDLEGVAZ However, this gene, like the two reference sequences, hits HNH chain A of the same Geobacillus virus E2 hit 5H0M_A in PDB, with 75.5% alignment, 99.19% probability, and E-value: 2.5e-11, with everything exactly as seen above for the two reference sequences, including the HNH endonuclease at position 76-124 (https://www.rcsb.org/structure/5H0M). Notably, Skinny gp 93 which is called HNH has got poor e-values Next is Glaske16 gene at 51656-52198 bp (draft gp 83); its sequence is below: MDTIWKPIPQDPTGLYLASQDGRILRKEYVIEKLQSHGHLYRRVMPEKIVKQCIKDRAPSHGVHPIIQMRSSTQYASTVERRVSSLIAAAWHGLPYEAGDRTAQNDWRIGFIDGDPSNVHADNLEWVSNQGVNTHHSHDFYYENLKAYRAQAAVETAESFLARYYSPDEIDWSTAERIAAZ This one too hits HNH endonuclease in phagesDB. HHPred shows it in PDB with 54.1% alignment, Probability: 99.76%, E-value: 6.2e-18, but notably, it does not hit the same chain as the ref chain (it hits 1U3E_M; https://www.rcsb.org/structure/1U3E) and no Zn+2 motif, but instead Mn+2 and Sr+2, but it also has the βα. What is your verdict on this gene in Glaske16 at 51656-52198 bp in view of the above? I am inclined to call it HNH endonuclease, except if the forum suggests otherwise. Next is Glaske16 gp 91 at position 54100-54426 bp. Has several hits to HNH in phagesDB. MPTNSKNGPRSRGRTGGKFERAKWRVLKANQICAHPDCRQLIDLDLKWPDPMSPTVNHIIPVKDLAWDDPLTYSVENLEPMHLVCNQRLGAGPRKKKPKHPQSRNWREZ This has a low e-value but hits the same chain as the ref sequence, and the zinc motif (https://www.rcsb.org/structure/5H0M ), and is called HNH endonuclease, and another hit at 4H9D_A (https://www.rcsb.org/structure/4H9D). What is your verdict on this gene in Glaske16 gp91 at 54100-54426 bp in view of the above? I am inclined but wary to call it HNH endonuclease because of the e-values, but again, it hits are the same as the Ref sequences; any suggestions? The next question is about the Glaske16 gp98 at position 56773-57150 bp. It has more than 60 hits to HNH endonuclease in phagesDB. Its sequence is below: MALAGEAKREYQRQWRANRRAAWFAGKACVRCGSDEDLELDHVDPTLKVTNAVWSWSQERRDVELAKCQVLCNACHKAKTISQTVITIGLKAYRHGTCSMYEHHRCRCGLCRLWARNKKRRQRAAZ It also hits the same hit 5H0M_A in PDB with the same everything as the reference sequences, and high probability (98%), alignment 52.4%, but with not as great an e value (0.000029). What is your verdict on this one? Finally, the Glaske16 gp117 at 60940-61320 bp. This gene has more than 70 hits to HNH endonuclease in phagesDB. What is your verdict on this one? Its sequence is: MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVEZ It also hits the same hit 5H0M_A in PDB (https://www.rcsb.org/structure/5H0M) with the same everything as the reference sequences Sisi gp 99 and Arianna gp 54, and high probability (98.05%), alignment 49.6%, but with not as great an e-value (0.000017). What is your verdict on this one? See details in attached file. Edited 09 May, 2023 18:55 1.54Mb

Link to this post | posted 04 May, 2023 23:11

fbaliraine

Thank you, Debbie! I will keep the gene with its tRNA overlap and push it over to QCer as you have suggested. I have tried searching for an article that would directly document evidence of overlaps between tRNA and protein-coding genes but was unsuccessful, although the Wright et al (2022) article (https://doi.org/10.1038/s41576-021-00417-w) has some documentation of overlaps between non-coding RNA (ncRNA) and protein-coding genes. Fred

Link to this post | posted 02 May, 2023 03:58

fbaliraine

In my previous post entitled “Is there any recent evidence of a tRNA overlapping a protein gene, even by a few bp?” (https://seaphages.org/forums/topic/5365/) I thought this question was settled. I want to delete the gene in phage Glaske16 at position 60940-61320 bp, but because several recent annotations have kept it, and it has more than 70 hits to the HNH endonuclease in phagesDB, I am seeking a second opinion on this. Its sequence is MQREYMRRWVANRRSAFFASKQCAMCGAGEELELDHIDPTKKVDHRIWSWTDARRSEELAKCQVLCASCHKKKTGEQWYANRSVSENAHHGTSRRYRKMKCRCGLCRLGNTNRSRALRQRHRVPVE Despite more than 70 hits to HNH endonuclease in phagesDB, this gene has low (<50%) coding potential in Genemark_S, and entirely no CP in Genemark_smeg, and TB. THIS GENE OVERLAPS 15bp WITH A tRNA CALLED BY ARAGORN v1.2.41 AND tRNA-SE v. 2.0. WITH AN INFERNAL SCORE OF 55.5. I am asking this question because of the statement from the Resource Guide entitled, “Predicting tRNA and tmRNA genes” (https://seaphagesbioinformatics.helpdocsonline.com/article-40): “It is highly unusual that a phage tRNA willi) Be encoded within an ORF called by Glimmer and GeneMark that has high coding potential, (ii) Be encoded on the opposite strand as a number of other phage tRNAs found in the same genome, (iii) Be encoded at a genomically distant location from the other tRNA genes in a genome. In general, violation of any of the three preceding conditions is sufficient for exclusion of a potential tRNA from an annotation (we have found a single high scoring tRNA that is not part of the rest of the large cluster, however this situation is very rare).” Whereas I am inclined to delete the protein coding gene and keep the tRNA since it is called by ARAGORN and tRNAscan-SE with a high infernal score (55.5), I also note that this tRNA is distant from other tRNAs, being 387 bp apart, which seems to violate caveat iii above. 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.” I realize that there may be exceptions though but wanted to be sure. Some M1 phages such as Reindeer & Iphrane7 do not have this gene but have the “Glu” tRNA (61306-61380 bp in Glaske16; see figures & DNA Master file attached), and we know that tRNAs tend to be conserved. Edited 02 May, 2023 04:16 346Kb 67Kb

Link to this post | posted 21 Jul, 2022 18:29
fbaliraine	Fabulous! Thank you Karen! Fred

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 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