Link to this post | posted 04 Apr, 2025 18:51

engstrom

debbie I think the best call is "ASCE ATPase", that is the term when you can't call it a RecA or provide more specificity. But you know you have an ATP-ase domain, yes? Seems to be missing the C terminus domain of a RecA. Is that what you are asking? debbie Hi Debbie, Thanks for your reply. We are pretty sure that we have structural homology to an ATPase domain, as it is the ATPase domain that HHPred finds. However, if we look at the slides for "How to call a RecA recombinase" we do see that to call an ASCE ATPase, our Pham must "Ha(ve) complete ATPase with Walker A, Walker B, and hydrolytic motif" That is the rub. We do not find the Walker A (almost there but missing the A/G) or the Walker B sequence motifs. We are also following Iyer et al 2004 Nucleic Acids Res Vol 32 p5260 which likewise indicates that any ASCE ATPase must have the Walker A and Walker B motifs. We are working up a poster on this problem, so maybe that will clear things up. Eric

Link to this post | posted 03 Apr, 2025 14:35

engstrom

Hi Folks, Monmouth College is annotating an EA1 phage genome, Symere, and we would like a check on a couple of Phams for which we have ascribed "Hypothetical Function". First is Stop-29,411. Lots of high probability HHPred hits, and everyone seems to favor a helicase or DNA helicase function, but we are not sure. You can view our detailed notes on Pecaan, but in brief the common functions seem to be nucleotide binding and a likely ATPase function. Not a RecA or ASCE ATPase as we can not identify WalkerA or WalkerB sequence motifs (and we have looked). Likewise with Stop-24,923, we can't find WalkerA (almost but just misses) or WalkerB motifs, so we cannot call either RecA of ASCE, yet this protein clearly has structural homology to these ATPases. We are happy to leave them as hypothetical proteins but this is going against the consensus so we thought to check. Many thanks for your thoughts!

Link to this post | posted 03 Sep, 2024 13:33

engstrom

Hi Folks, However, what Eric Engstrom should have written would be "we switched from Fisher Brand Peptone to Bacto Peptone." I should read my notes before posting anything. We did indeed try the agar switch first, but as I review this, that does not appear to have been the fix. The subsequent shift to Bacto Peptone is what saved our bacon. Heartfelt apologies for disseminating inaccurate information. I was traveling and relying on my memory, rather than my notebook. Never a good idea. Eric

Link to this post | posted 06 Oct, 2023 15:12
engstrom	Thanks Everyone! We MAY have achieved a breakthrough yesterday by using Millipore water. Haven't run a plaque assay yet, but the appearance of the TA is encouraging. Looks like our DI system may need some maintenance. I'll report on the assays after next week. Eric

Link to this post | posted 05 Oct, 2023 14:52

engstrom

Hi Community, We have been stopped in our tracks here at Monmouth, by a very strange issue. Our PYCa top-agar is useless as our CaCl2 is consistently precipitating. We made a new 1M stock of course. We made new dextrose as well. We played with our autoclaving times. And we are not having any trouble with plate agar. At a loss. My question–can we use top-agar for plaque assays if we omit the CaCl2? This would seem to be the quickest route to solving our problem and we will make a test of it soon. Other suggestions? Thanks!

Link to this post | posted 18 Aug, 2023 00:05

engstrom

Hi Folks, I am looking at a possible (but I am increasingly thinking unlikely) tRNA in Enygma at roughly 88430-88539. Infernal score is low, but just misses the cutoff at 29.8. Size of the tRNA is within 90 nt, but only just. Aragorn, Aragorn 1.2.38 and tRNAscan-SE all draw different conclusions about the anti-codon. Aragorn 1.2.38 wants to put an intron in, which makes the least sense of anything to my mind. There is no syntenic tRNA annotated at this location in any of the most closely related genomes (Bordeaux, SaltySpitoon, PumpkinSpice). Student annotator took it out. I am leaning toward agreeing, but would value a third opinion.

Link to this post | posted 04 Jun, 2023 21:35

engstrom

I am looking at Stop-8213 in Enygma, a BE cluster phage. HHPred finds significant homology to L7/L12 subunit proteins of the 50S Ribosome in two bacterial species. There is however no detected sequence homology to back this up. Homology occurs across a significant chunk of the Pham, but not its entirety. aa sequence–MERIEIVQQVGNYFPDTKIADALRFAGDIDRGFARKMEDAQYKAEDEKYEYAEKRYNEGVKAGKESVRTTFDPQVVEATIWATTNFTDRDLTRKITCIKVLRDKFRPLGLIEAKTIMDMIKPLGTTDTLDWPKIVEAEKDSPELATQSALNALRNKLTNNEVARESIDSEESESNDYSTCPCGCGGMA https://www.rcsb.org/structure/1DD3 I suspect that we need more evidence, but thought it worthwhile enough to post.

Link to this post | posted 13 Apr, 2023 14:41

engstrom

tRNAScan-SE v 2.0 identifies a Ser tRNA in Huwbert (53324-53234). 90 nucleotides, so a tad big for comfort. Aragorn v 1.2.41 identifies a Ser tRNA in Huwbert (53233,53324). 92 nucleotides–even worse. But if we set Aragorn search parameters to allow introns, we instead get a Val tRNA (53233,53324) 88 nucleotides. We are a bit stuck on 1.) is this a legitimate tRNA call (Triscuit did not call it), and if so, do we allow the intron?

Link to this post | posted 09 Apr, 2023 14:57
engstrom	Thanks–that is really quite helpful. We'll leave it a primase/helicase and move along, but I do think that this gene warrants a deep-dive. I'll put it on my list of potential student projects.

Link to this post | posted 06 Apr, 2023 22:49

engstrom

I could use some thoughts on an interesting gene in Huwbert, called stop codon is 58,402. This is syntenic to a Pham called as RecA-like DNA recombinase in Triscuit. But I am not so sure. Nucleotides 335-568/9 find homology to RecA-like recombinases, but only to the AAA-ATPase domain (HHPred). Likewise HHPred picks up other proteins that have an AAA-ATPase in the same stretch. From nt 1-312, there is strong predicted structural homology to Replication Protein B Primase, a function not in the approved function list. Not completely sure where to go with this thing. I could call a DNA primase/helicase, but do I actually have evidence for the helicase function, or just for an AAA-ATPase. A primase would not need an ATPase, so I rather suspect the helicase is there. Anyway, I'd love some feedback. Thanks.