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

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

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Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
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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)

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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

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