Dear All,
To add my voice to those who wrote crystallization artifacts happen - we
just witnessed one today. A postdoc from another lab tried crystallizing
a protein for months. Today, during data collection from his poorly
diffracting crystals, one of our guys (Dr. Porebski) scaled the data and
checked if there is a structure with the same Sp Gr and unit cell
parameters in the PDB. And there was one - 4ZNZ (Escherichia coli
carbonic anhydrase (YadF) in complex with Zn - an artifact of
purification), deposited by our group two years ago. Quick molecular
replacement showed that it is indeed the protein we had in the beam today.
The protein looked good on the SDS gel. The band for YadF was definitely
less than 1% of the protein sample (less than one percent!!! if the gel
would not be overloaded, nobody would see the band), but it still
crystallized. We speculate that YadF from Escherichia coli was
co-purified with the target protein due to relatively strong
protein-protein interactions despite multiple purification steps.
We did not use ContaMiner, though. Instead, we just used the search of
the unit cell parameters in the PDB, which is much faster (but works
only if the structure of this particular artifact in the same SpGr is in
PDB! otherwise, one should use ContaMiner or similar service). This
feature is implemented in HKL3000, but it can also be done quickly on
the PDB webpage. The procedure, as well as cases with other
crystallization artifacts, is described in:
Protein purification and crystallization artifacts: The tale usually not
told. (2016) Protein Sci. 25: 720-733
Today's example clearly shows that depositing these artifacts can
greatly help others (it took just a few minutes to realize we had an
artifact). Therefore, I would like to encourage everyone to deposit
their artifacts to the PDB, especially if these artifacts were
crystallized with unit cell parameters not reported previously. An
increase in the size of the library of crystallization artifact
structures deposited to the PDB can make troubleshooting of new
artifacts much easier, and save much effort for those who are new (or
not very new!) to such cases.
With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908
On 11/23/2017 02:35 PM, 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
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