Open LizbeK opened 4 years ago
mattodd
commented
4 years ago Hi @LizbeK . Thanks for the update. So am I right in thinking that despite the technical challenges you've had, you're nevertheless confident that none of the fragments we shipped have bound? i.e. we should consider them "inactives". It's not as though we ought to be waiting for further analysis and/or experiments?
LizbeK
commented
4 years ago @mattodd @danaklug @fidiris Consider them "inactives". Sorry.
mattodd
commented
4 years ago OK, @fidiris and @danaklug are going to collate what we know and try to make sense of the results, which are quite surprising. One last query, relevant to the question we were discussing about whether there was non-overlapping substrate bound in the crystal. The assay is done in such a way that if substrate needs to be bound (i.e. ATP or amino acid or sugar) that it could be bound? i.e. it matters that we're manipulating protein in this assay that could have non-overlapping substrate if that is needed. Apologies if this is a naive question, I don't have details of the assay to hand.
fidiris
commented
4 years ago Myself and @danaklug have now created a new page on the wiki to summarise the fragment analogues which were shipped for evaluation back in December. Hopefully having all the structures in one place will make it easier to understand the rationale behind why those compounds were synthesised.
The results for cluster 6 were particularly suprising for us. Having looked at the fragment analogues synthesised, a lot of them are structurally very similar to the original hits.
Would it be worth sending samples of the original hits for next rounds of screens as controls?
drc007
commented
4 years ago Would it be worth sending samples of the original hits for next rounds of screens as controls?
I agree
bendndi
commented
4 years ago Would it be worth sending samples of the original hits for next rounds of screens as controls?
If you're also able to get a new sample of the original hits I'd suggest testing that too.
The page on the wiki looks good. You may have already done this but is there also a plan to have all this info collated in a single "live and updatable" file, ideally an sdf file or csv/ smiles file for use in datawarrior / excel ?
LizbeK
commented
4 years ago I would actually suggest "new / fresh" sample of the original hit on the same echo plate the compounds are going to be on; diluted and pipetted by the same person who prepares the rest of the samples. In that case it will be a good controle all the way through the experiment, which includes handling, shipping, storage, echo transfer, and the actual soaking.
fidiris
commented
4 years ago If you're also able to get a new sample of the original hits I'd suggest testing that too.
Yes, so I've synthesised one of the hits for MurE (OSA_000005) and with the next MurD shipment I'm in the process of making or buying the original hits. We will do the standard characterisation (1H and LCMS) and check the purity before shipping for commercial compounds.
There is currently a google sheet that myself and @danaklug have been updating with our compounds. Is that what you mean? or do we need another file?
LizbeK
commented
4 years ago OK, @fidiris and @danaklug are going to collate what we know and try to make sense of the results, which are quite surprising. One last query, relevant to the question we were discussing about whether there was non-overlapping substrate bound in the crystal. The assay is done in such a way that if substrate needs to be bound (i.e. ATP or amino acid or sugar) that it could be bound? i.e. it matters that we're manipulating protein in this assay that could have non-overlapping substrate if that is needed. Apologies if this is a naive question, I don't have details of the assay to hand.
To answer @mattodd earlier question, for the XChem screen the crystals are prepared in the absence of any substrates and the soaking is also done in the absence of substrates.
drc007
commented
4 years ago Hi,
Did you get crystals for all samples? Or did you get some compounds causing melting of crystals?
On 7 Feb 2020, at 14:19, Lizbé Koekemoer notifications@github.com wrote:
Update 07/02/2020: I have manually looked through all the data sets focusing on the Cluster 6 and 15 pockets and couldn’t see anything bound in these sites. We are currently dealing with some challenges with the data analysis of these specific datasets with the PanDDA software. Due to the low symmetry of the crystals (P1) the analysis takes very long and we get a lot of false results due to subtle rearrangements in certain side chains and loops which we don't understand have a good handle on. This is not the same conformational change observed between the apo and substrate bound forms. We are looking at clustering the data to see whether we can pin down some of these more subtle rearrangements. The good news is PanDDA version 2 is scheduled for release soon, which we already know will speed up the analysis process a lot.
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LizbeK
commented
4 years ago Hi, Did you get crystals for all samples? Or did you get some compounds causing melting of crystals? …
@drc007 I had no melting of crystals in this batch. I attach the relevant columns of the database file. The column you want to look at is MountingResult (column F). The comments looks something like this: OK:Clear:Normal
First comment is either OK (mounted) or Fail (not mounted). All the soaked crystals were mounted. Second comment describes what happened to the compound added to the drop. Clear (can't distinguish it from crystal drop), Phase separation (not mixing with crystal drop), Precipitated out, Crystalline (crystallized out), No comment (same as normal) Third comment describes what happened to the crystal. Normal, Cracked, Melted, Jelly, No comment.
2020-02-12_Follow-up 1 soakDB file.xlsx
I noticed that 15 of the crystals didn't diffract (column G-I). I highlighted them in yellow.
Update 07/02/2020: I have manually looked through all the data sets focusing on the Cluster 6 and 15 pockets and couldn’t see anything bound in these sites. We are currently dealing with some challenges with the data analysis of these specific datasets with the PanDDA software. Due to the low symmetry of the crystals (P1) the analysis takes very long and we get a lot of false results due to subtle rearrangements in certain side chains and loops which we don't understand have a good handle on. This is not the same conformational change observed between the apo and substrate bound forms. We are looking at clustering the data to see whether we can pin down some of these more subtle rearrangements. The good news is PanDDA version 2 is scheduled for release soon, which we already know will speed up the analysis process a lot.