Hi Radu thank you for your nice message - it is indeed unclear how often these cases happen. From our side it was nice to hear that people acknowledged the usefulness of our tool - which can also be used to rule out (known) contaminant crystals. Given that our ContaMiner will (by its design) not be cited much (contaminants don't get published), such positive feedback is our source of happiness.
With best wishes, Stefan On 27 November 2017 at 12:35, <r...@mrc-lmb.cam.ac.uk> wrote: > Hi Stefan, > > I also owe you an apology for my bad choice of words. One can debate about > usefulness, but Pavel is right, the software is not "bizarre". And, as > several > people pointed out, it certainly addresses a need (which I didn't know > about). > Mea culpa. > > Best wishes, > > Radu > > > > 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 > >> > > > > > -- > 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 > >
