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
