Dear Gerard I am really sorry that my badly formulated 'final word of warning' has made you and others spend much time for composing well-formulated replies. I was at PX1 yesterday, and Leo reminded me of the issue, hence I included it into my message. You are absolutely right that reports of anomalies should go to the developers - however the issue here is already known (as shown in the STARANISO Warning message) and cannot be solved by us at the ContaMiner level. Given the popularity of Staraniso, I just wanted to inform our users that this particular issue is propagated into the ContaMiner results. I should have worded it more carefully.
Many thanks to Leo and Pierre for helping explain! With best wishes Stefan On 24 November 2017 at 13:07, Gerard Bricogne <g...@globalphasing.com> wrote: > Dear Radu, > > I would not want to take undue advantage of this already > voluminous thread, but your PS takes us into a different direction, > namely the whole myth-ridden topic of "weak data" - but that will be > for another thread, another time ;-) . > > > With best wishes, > > Gerard. > > -- > On Fri, Nov 24, 2017 at 01:02:08AM -0000, r...@mrc-lmb.cam.ac.uk wrote: > > Hi Leo, > > > > I agree that the horror beamline stories you describe are far too common. > > Unfortunately, they start earlier, in the wet lab or even before. > Exactly the > > same attitude (careless construct design, crystallising whatever "dirty" > > samples, not bothering optimising cryoprotection and so on) leads to > wasting a > > lot of resources, including synchrotron time. In some cases, as people > pointed > > out, problems such as contaminations (and even more so anisotropic data, > for > > the matter) are unavoidable. But too often, as we all know, it's simply > bad > > practice, lack of training etc. Web servers can only help up to a > point... > > > > Best wishes, > > > > Radu > > PS: At least, one day, maximum-likelihood refinement programs will deal > with > > weak data satisfactorily :-) Nobody likes to throw data away. > > > > > > > Dear all, > > > > > > to join Pierre's comments on what 'strange' things happen at the > beamlines... > > > yet not too strange for (too) many people: huge screening of salt > crystals, > > > complete data collection of dramatically low resolution data, full > power > > > coupled with 360Deg data collection etc. etc. etc. We do unfortunately > see too > > > many 'blind shots, deal with it later, and move on' experiments that it > > > becomes depressive. I personally do not see why we would close our > eyes to > > > servers and/or data analysis tools that could help you think less, or > better > > > say help you understanding what is eventually happening with your data. > > > > > > Cheers, leo > > > > > > - > > > Leonard Chavas > > > - > > > Synchrotron SOLEIL > > > Proxima-I > > > L'Orme des Merisiers > > > Saint-Aubin - BP 48 > > > 91192 Gif-sur-Yvette Cedex > > > France > > > - > > > Phone: +33 169 359 746 > > > Mobile: +33 644 321 614 > > > E-mail: leonard.cha...@synchrotron-soleil.fr > > > - > > > > > >> On 24 Nov 2017, at 00:23, Edward A. Berry <ber...@upstate.edu> wrote: > > >> > > >> My 2 cents worth: > > >> I think contaminer is an extremely useful service. I may be a sloppy > > >> biochemist, > > >> but I am not the only one. There are multiple structures in the > database of > > >> say > > >> bacterioferritin or AcrB that were solved from crystals that were > supposed > > >> to > > >> be something else. I remember in a discussion with the organizer of my > > >> session > > >> at a Gordon conference, she excitedly announced that there would be > > >> preliminary > > >> crystallographic data on respiratory Complex I. But by the time of the > > >> conference > > >> the authors discovered they had crystallized something else. And the > > >> beautiful crystals > > >> of Paracoccus Complex II (from Doug Rees's lab?) that graced the > catalog of > > >> Hampton Research (And I believe were part of the basis for the first > > >> membrane > > >> protein screen) never saw publication. The authors of > > >> http://www.sciencedirect.com/science/article/pii/S0304416506000894 > > >> certainly feel there is a real problem. Some proteins crystallize > readily > > >> even when > > >> present as minor contaminants. And some protein complexes become more > > >> heterogeneous > > >> if over-purified due to partial loss of loosely-bound subunits. > > >> Most of my career I've worked with high-abundance natural-source > proteins. > > >> During a recent foray into the realm of overexpressed proteins, my > group > > >> has > > >> crystallized (and solved) at least a half dozen wrong proteins from E. > > >> coli. > > >> I spent months on one of these (ATCase in Rhomb sg with low-level > > >> obverse/reverse > > >> twinning that caused it to sometimes index as P3) Then solved the rest > > >> rapidly > > >> by checking the closest several hits with nearest-cell. All of these > E.coli > > >> proteins > > >> were already present in the PDB. I wonder how many were from > accidental > > >> crystals. > > >> And now bacterioferritin (this time from M. smegmatis) keeps coming > back to > > >> haunt us. > > >> > > >> I would say any time with a new crystal when a molecular replacement > > >> unexpectedly fails, > > >> and even before you start to collect heavy atom or selenomet data, it > would > > >> be worth > > >> to submit to nearest-cell and contaminer. I would be more likely to > question > > >> the > > >> utility of an anisotropy correction server, given that modern > > >> maximum-likelihood > > >> refinement programs can deal with weak data satisfactorily (speaking > from > > >> ignorance- I'm sure supporting evidence and examples exist, I just > haven't > > >> bothered to look them up. And I know my colleagues here at Upstate > have > > >> used > > >> anisotropy correction to good effect with a difficult problem- I hope > they > > >> weren't using filled-in maps!) > > >> eab > > >> > > >> On 11/23/2017 03:24 PM, Tristan Croll wrote: > > >>> Dear Radu, > > >>> > > >>> I think this is a little harsh. Biology is a fabulously messy thing, > and > > >>> very prone to doing the unexpected. See the excellent paper by > > >>> Niedzialkowska et al. at > > >>> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815408/#!po=13.6905 > for some > > >>> examples. Sometimes unexpected things (which just happen to have a > similar > > >>> size to your target) carry through all the purification steps - I > remember > > >>> having terrible trouble isolating his-tagged IGF-I (not for > > >>> crystallization) from Sf9 lysates due to a cathepsin-like protease > that > > >>> stuck doggedly to the Ni-NTA column even under 8M urea, yet > co-eluted in > > >>> imidazole. Even if contaminant proteins are barely visible on your > SDS-PAGE > > >>> gel, if they crystallise easily and your target doesn???t... all > these > > >>> things and many others have happened, and have undoubtedly driven the > > >>> occasional poor grad student to the brink of giving it all up. > > >>> > > >>> I guess in these days of relatively cheap and ubiquitous mass spec > it may > > >>> make sense to sacrifice a crystal to trypsin digest and MS/MS > sequencing > > >>> just for peace of mind, but in the average case I think that???s > likely to > > >>> be overkill. Shooting crystals at a synchrotron is now very routine, > so I > > >>> think it makes perfect sense to provide a computational check for the > > >>> (hopefully rare) surprise case. > > >>> > > >>> Best regards, > > >>> > > >>> Tristan > > >>> Tristan Croll > > >>> Research Fellow > > >>> Cambridge Institute for Medical Research > > >>> University of Cambridge CB2 0XY > > >>> > > >>> > > >>> On 23 Nov 2017, at 19:35, r...@mrc-lmb.cam.ac.uk > > >>> <mailto:r...@mrc-lmb.cam.ac.uk> wrote: > > >>> > > >>>> Dear Stefan, > > >>>> > > >>>> Just a couple of thoughts: > > >>>> > > >>>> - first of all I think that Gerard is absolutely right, it would > have > > >>>> been > > >>>> nice to raise such issues first with the developers. In my > experience, > > >>>> Staraniso does a fantastic job if used correctly. > > >>>> > > >>>> - but if you're OK with public trials, may I ask: why on Earth would > > >>>> anybody > > >>>> need ContaMiner? Are you trying to offer some sort of computational > cure > > >>>> for > > >>>> sloppy biochemistry? There is zero point in crystallizing crap > samples, > > >>>> sorry > > >>>> to say this. In my 17 or so years in Strubi I've never heard of > anybody > > >>>> crystallizing a "contaminant", being it a purification tag or > whatever. > > >>>> > > >>>> I suppose this might have happened to somebody you know, hence the > > >>>> motivation > > >>>> to spend time on the bizarre ContaMiner. Which is a pity, a silly > outcome > > >>>> would only teach people to do their job (or train their robots) > properly. > > >>>> > > >>>> Best wishes, > > >>>> > > >>>> Radu > > >>>> > > >>>> -- > > >>>> Radu Aricescu > > >>>> MRC Laboratory of Molecular Biology > > >>>> Francis Crick Avenue > > >>>> Cambridge Biomedical Campus > > >>>> Cambridge CB2 0QH, U.K. > > >>>> tel: +44-(0)1223-267049 > > >>>> fax: +44-(0)1223-268305 > > >>>> www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu- > aricescu > > >>>> > > >>>>> Dear Stefan, > > >>>>> > > >>>>> Regarding your final paragraph: your server carries a warning > > >>>>> with the exact wording: > > >>>>> > > >>>>> "Submitting StarAniso files can give you suspicious results. Use > > >>>>> with care!" > > >>>>> > > >>>>> It seems rather regrettable that you are posting such a public > > >>>>> warning without ever having contacted the STARANISO developers > about > > >>>>> your observations, nor giving any information about what you call > > >>>>> "suspicious" or what the "care" you recommend would consist of. > > >>>>> > > >>>>> We have taken a great deal of care ourselves in developing the > > >>>>> program and offering it to the community through a server, and the > > >>>>> least we would have expected is that any pattern of "suspicious" > > >>>>> results would be referred to us so that we could investigate them. > > >>>>> There may be some assumptions made in MoRDa that we are not aware > of, > > >>>>> that might be incompatible with assumptions made in STARANISO - who > > >>>>> knows? Or it could be that some particularly badly collected > datasets > > >>>>> are made to look worse after their anisotropy analysis. > > >>>>> > > >>>>> Could we discuss your observations, and what it is exactly that > > >>>>> you call "suspicious", before they end up being referred to in > such an > > >>>>> uninformative manner as some sort of "Government Health Warning"? > > >>>>> > > >>>>> I think that would be nice :-) and we would be only too keen to > > >>>>> take whatever extra "care" is needed ourselves. We would all learn > > >>>>> something. > > >>>>> > > >>>>> > > >>>>> With best wishes, > > >>>>> > > >>>>> Gerard. > > >>>>> > > >>>>> (on behalf of the STARANISO developers) > > >>>>> > > >>>>> -- > > >>>>> On Thu, Nov 23, 2017 at 05:02:39PM +0100, Stefan Arold wrote: > > >>>>>> Dear Community, > > >>>>>> > > >>>>>> A quick message to announce the following two new features on our > > >>>>>> ContaMiner web server for the automated detection of unwantedly > > >>>>>> crystallised contaminants ( > > >>>>>> https://strube.cbrc.kaust.edu.sa/contaminer/submit) > > >>>>>> > > >>>>>> 1) online visualisation of 2FoFc and FoFc maps. In cases of > positive > > >>>>>> results, the ???UglyMol??? tab allows to inspect 2FoFc and FoFc > maps > > >>>>>> directly > > >>>>>> in the web browser. Thi > > >>>>>> > > >>>>>> 2) life-update. Previously, results were sent to you once all > ~2000 MR > > >>>>>> jobs > > >>>>>> were finished. Now, the individual results for each potential > > >>>>>> contaminant > > >>>>>> will appear as soon as they are finished. This feature should > > >>>>>> substantially > > >>>>>> shorten the time for identifying positive results (i.e. > contaminant > > >>>>>> detected), which are terminated faster than negative ones. > > >>>>>> > > >>>>>> 3) custom contaminants. In the ???Advanced??? tab, users can > upload own > > >>>>>> PDB > > >>>>>> files (more than one is possible) to be included as search > models. This > > >>>>>> feature can be used to include PDB files from your lab bench > > >>>>>> neighbour???s > > >>>>>> project to test for potential lab internal contaminations (through > > >>>>>> bacterial contamination or through mix-up of plasmids or glycerol > > >>>>>> stocks). > > >>>>>> This feature could also be ???abused??? as a means to use the > MoRDa > > >>>>>> pipeline > > >>>>>> to > > >>>>>> run molecular replacements with template structures that are not > yet > > >>>>>> deposited in the PDB; for example to run molecular replacement and > > >>>>>> initial > > >>>>>> refinement for liganded or complexed versions of an unpublished > > >>>>>> structure. > > >>>>>> This might be particularly interesting for crystallographers away > from > > >>>>>> their usual home software environment (e.g. at the beamline). > > >>>>>> > > >>>>>> Finally, a word of warning ??? Staraniso files might give false > > >>>>>> positives if > > >>>>>> they have large anisotropic cuts. > > >>>>>> > > >>>>>> Keep your crystals clean! > > >>>>>> > > >>>>>> With best wishes > > >>>>>> > > >>>>>> The ContaMiner Team > > >>>>> > > >>>>> -- > > >>>>> > > >>>>> =============================================================== > > >>>>> * * > > >>>>> * Gerard Bricogne g...@globalphasing.com > > >>>>> <mailto:g...@globalphasing.com> * > > >>>>> * * > > >>>>> * Global Phasing Ltd. * > > >>>>> * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * > > >>>>> * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * > > >>>>> * * > > >>>>> =============================================================== > > >>>>> > > > > > > > > > > > > -- > > Radu Aricescu > > MRC Laboratory of Molecular Biology > > Francis Crick Avenue > > Cambridge Biomedical Campus > > Cambridge CB2 0QH, U.K. > > tel: +44-(0)1223-267049 > > fax: +44-(0)1223-268305 > > www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu >
