Dear Stefan,
Thank you for your message. I am sure that we will figure out
what caused the spurious results described by Pierre, now that we know
about them ;-) .
Perhaps my reaction to your message was a little too loaded with
tension, and if so I regret it. The fact is that we are encountering
much "mental viscosity" in our discussions about a simple framework
for anisotropic statistics, which makes it hard not to become a bit
defensive at times.
It was nice to read that your ContaMiner server is so highly
appreciated!
With best wishes,
Gerard.
--
On Fri, Nov 24, 2017 at 03:12:50PM +0100, Stefan Arold wrote:
> 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
