Link to this post | posted 15 Feb, 2022 16:34

fbaliraine

During the recent workshop, two the programs DNABIND and DNA Binder were mentioned for predicting DNA Binding Proteins. We have found several genes that have been predicted by both programs to be DNA binding proteins (varying strengths), but do not necessarily have strong HHpred alignments to DNA binding. Current BLASTp hits in NCBI and phagesDb are “Hypothetical Proteins.” However, these genes also appear to be in either an operon or in the syntenic region with other DNA binding proteins, such as DNA methylase, translocase, resolvase, and specific-DNA-binding proteins. Can we call these genes the general name of “DNA binding protein” based on the two programs and the strong possibility of the operon or in syntenic region? In general, is it possible to call DNA binding proteins based on these two programs alone? Two examples from the P1 phage Dynamo are gp 44 (start/stop: 31954-32103) and gp 51 (36150-3642. See attached file. Thanks! Fred 603Kb

Link to this post | posted 21 Jul, 2021 22:21

fbaliraine

Hi Debbie, Thank you for helping put this to rest. We have been using PDB, PfamA, CDD, and Scope! We have not been including the Uni-Prot-Swiss Protein-viral database. Now when I exclude scope like you did and use Uni-Prot-Swiss Protein-viral, I can see hits in HHPred to minor tail proteins! Previously there was no significant hits to anything similar. Perhaps it might help to include the use of Uni-Prot-Swiss Protein-viral rather than or besides Scope in the resource guide for HHPred. Case closed! I will go ahead and name them minor tail proteins. Thanks again! Fred

Link to this post | posted 19 Jul, 2021 23:21

fbaliraine

Genes 5, 6, and 7 of phage Mach and gp 6 in phage Duplo (as per current naming in phamerator before annotation) are showing synteny with minor tail proteins in phamerator, and also hitting minor tail proteins in phagesDb and NCBI (q1:s1, 99-100%). However, none of them have any significant hits to minor tail proteins in HHPred. I have tried checking the forum to see if anyone previously asked a specific question about minor tail proteins occurring this far upstream of the tape measure protein, but I have not seen such a direct question or answer in this regard. What I can see from previous posts is that minor tail proteins should typically come after the tape measure (downstream) or just before it (upstream) in some clusters. https://seaphages.org/forums/topic/4872/ and https://seaphages.org/forums/topic/4836/ The official functions list states that “If you have significant hits to either collagen-like or glycine-rich proteins, and are in the syntenic region of minor tail proteins, you can call them minor tail proteins.” But none of this is seen in HHPRED in phage Mach and Duplo. Of note, phages Mach and Duplo are Siphoviridae, and Siphoviridae genes are expected to occur in the following order: Terminase small subunit – Terminase large subunit – Portal – Protease – Capsid – Head-Tail Connectors/adaptors – Major Tail Subunit/major tail protein – Tail assembly Chaperones –Tape measure Protein – Minor Tail Proteins (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069273). Thus, synteny, per this Pope et al. 2013 does not seem to support minor tail protein in this region (see pg 4 under “Virion structure and assembly genes” in this paper). I need to be sure whether something has changed since this article was published about 8 years ago. Based on the above, I am inclined to call them “NKF” rather than “minor tail proteins,” but since there are several hits to “minor tail proteins” in phagesDB as well as having synteny in phamerator, I am seeking the forum’s informed clarification. Below is a sample sequence, Mach gp 6, is anyone wants to cross-check by BLAST in phagesDB and HHPred: MANIGIVSDADTLVLWKGRDFKWSFENLDENRQPVDFPDGSLFIELQTGGEHNARQRVTITGATGGTYAFDILGETTPPIDYNDVSENPQGLPGDITEALEAAAGVGNVEVYPTLLQPSWILNFNLNSGKPLTEQLVNTINKTANDFFDTFEQLMGVDVSMTVTDALNFQLKVTSRRSFDEVGVVTFAVDVTGTAVKNFFNAVSGLVGAVNTVNVDFYWNRVYEIEFVGELANQPIEAILPDASNLTGYNPWITVEVIDLGKERLTIWPFIIDGTEATIKVESEEADLIPERTVWQLVFLPDGEPAGGDPITYGRVTRLGDZ I have also attached a searchable phamerator map for phage Mach that demostrates what I am seeing. Thanks! Fred Edited 19 Jul, 2021 23:34 1.05Mb

Link to this post | posted 10 May, 2020 19:38
fbaliraine	Great! Thanks Debbie! Fred

Link to this post | posted 10 May, 2020 18:13

fbaliraine

Dear phage hunters, since the very first auto-annotation till now, we’ve had trouble getting NCBI blast data for Shida Gene 7 when we do Blast through DNA Master, but can get the blast data when we do the BLASTp directly on the NCBI website, as well as in PhagesDb. This gene is present in Vorrps, YungJamal & Krili in phamerator and had q1:s1, 100% with phage Krill in phagesDb; Original GeneMark call @bp 2236; SSC: 2144-2236 rev; CP: Yes; SCS: Both; ST:SS; BLAST-Start: Krili, gp 7, phagesDb, q1:s1, 100%, 8e-12; Gap:4 bp overlap. TMHMM and SOSUI analysis confirmed this to be a membrane protein, with one transmembrane domain. Since we have tried several times without success getting Blast data for this gene via DNA master and its needed for submission of the final files, what would you advise us to do? This is the only gene in Shida that has not budged! Attached are snapshots of the Frames, Coding potential profile, phamerator snapshot, results (actually no result) when Blasted via DNA Master, PhagedDb BLASTp results, NCBI BLASTp results (when done directly via the website), TMHMM & SOSUI analysis results. Please advise, because we have tried to get blast data for this gene directly via DNA Master as required, but all in vain. Fred Edited 10 May, 2020 18:17 448Kb

Link to this post | posted 09 May, 2020 02:46
fbaliraine	Thank you Debbie! Fred

Link to this post | posted 07 May, 2020 00:14

fbaliraine

Dear Phage hunters, I Need help deciding on the function of MrMiyagi gene 97 (gene 100 in phamerator). It is an Orpham. BLASTp in NCBI & phagesDb hits holliday junction resolvase with phage Fowlmouth, q59: s7, coverage 52%, pecent identity 96.97%, e-value 1e-25 (https://blast.ncbi.nlm.nih.gov/Blast.cgi). However, HHPred data hits at DNA Helicase, PDBe, Thermus thermophilus, d1ixsa_, 28.92%, 92.41% as well as RNA POLYMERASE SIGMA FACTOR CNRH; TRANSCRIPTION, ECF-TYPE SIGMA and on RuvA_C ; RuvA, C-terminal domain.https://toolkit.tuebingen.mpg.de/jobs/MrMiyagi_gp97 The hit at Holliday junction DNA helicase ruvA/RuvB(E.C.3.6.1.3) has a probability of 78.61%. I am therefore conflicted on what function to settle for: holliday junction resolvase, DNA Helicase or RNA polymerase sigma factor? There was a recent post about sigma factor but I dont see it. Fred Edited 07 May, 2020 00:18 265Kb

Link to this post | posted 06 May, 2020 21:59
fbaliraine	Thanks a lot Welkin! Fred

Link to this post | posted 05 May, 2020 19:23

fbaliraine

Having trouble deciding on current Genes 42 and 43 in Phage MrMiyagi. Attached, please find snapshots of RBS scores, Frames, Coding potential, and Phamerator maps. These two gens apparently have overlapping ORFs. No CP seen in GeneMark-smeg & TB, but strong coding potential for both in GeneMarkS. Start for gp 43 (45 in pharmerator) called by GL & in Cuke & Fowlmouth; staterator; SSC: 35734; Z= 1.676, F= -6.512; spacer distance 16; with 6 bp overlap; we were selecting the start at 35716, Z= 2.487, F= -5.050, 26 bp overlap with gene 42 (44 in phamerator); with a shorter spacer distance of 5, but realize this gene 43 ORF is continuous from 35449 bp (Z= 2.018, F= -5.536; the next start is with a better RBS at 35572 bp with Z= 2.673, F= -3.504). The short gene 43 (45 in phamerator) apparently currently has only 3 members in the pham 32826; compared to 44 which has 61 members (pharm 40615). Start for gene 42 (44 in phamerator) is inside CP plateau, hence not covering all CP, but it’s the 1st one; see CP figure attached. Since there is no break/stop codon in the ORF for gene 43(45 in phamerator) and there’s strong RBS scores for the 1st and 3rd starts which are overlapping gene 42 (44 in phamerator) is there no chance that this is the same gene? Should I delete 42 (44 in phamerator) and take the start at 35572 bp with Z= 2.673, F= -3.504, or should I just overlook this, keep gene 42 (44 in phamerator) and take the start called by GL at 35734; z= 1.676, F= -6.512 and in Cuke & Fowlmouth 6bp overlap, or the one I am considering with a better RBS score at 35716, Z= 2.487, F= -5.050, 26 bp overlap? Please advise. Edited 06 May, 2020 16:22 734Kb

Link to this post | posted 05 May, 2020 16:29
fbaliraine	Great insight! Thanks Debbie! Fred