Ice rings...

From: Francis E Reyes
Date: 11 October 2011 16:16


All,


So I have two intense ice rings where there appear to be lattice spots in between them.

I understand that any reflections that lie directly on the ice ring are useless, however, how do software programs (HKL2000, d*Trek, mosflm, XDS) deal with these intermediate spots?

It would seem to me that employing a 'resolution cut off' just before the ice ring (on the low resolution side) would be improper, as there are spots on the high resolution side of the ice. (see enclosed .tiff)


In fact, how do these programs deal with spots lying on ice rings? Are they rejected by some algorithm by those programs during integration, or is it up to the scaling/merging (by SCALA for example) step to deal with them?

Thanks!

F



---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder







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From: Bruno KLAHOLZ


Dear Francis,

the spots will be excluded individually based on the inhomogeneous background, so you don't need to apply a resolution cutoff.
However, once you have determined and refined your structure it may be worth predicting the intensity of these spots and put them back for map calculation,
this might avoid gaps in your map corresponding to inter-atom distances for which data are missing in the resolution range of the ice rings; as long as this is done only for a relatively small set of reflections there is not much risk of introducing a bias here.

HTH,

Bruno




-
Dr. Bruno P. Klaholz
Department of Integrated Structural Biology
Institute of Genetics and of Molecular and Cellular Biology
IGBMC - UMR 7104 - U 964
1, rue Laurent Fries
BP 10142
67404 ILLKIRCH CEDEX
FRANCE



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From: Edward A. Berry


If the ice rings are really sharp, they trigger the bad
background rejection in denzo/HKL2000. To reject more spots,
increase the "reject fraction 0.7" parameter to something
greater than .7. This rejection is on a spot by spot basis,
so spots with good background between the rings should not
be affected. During integration, if you are monitoring
the process with Xdisp, you will see the rejected spots
turn red and/or disappear. To verify they are being
rejected by background fraction, try again with
"reject fraction .3" and see if they stay green/yellow.

If the ice ring is broad compared to the integrating box,
it shows up as a high, slanting baseline and the normal
baseline correction procedure is valid, but sigma will
be higher than for a spot on a white background.

Francis E Reyes wrote:

---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

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From: Dr. Thayumanasamy Somasundaram

Francis,

I would like to bring your attention to our paper in Acta Cryst D Volume 66 (6), 741-744 (2010) where we deal with spots under the ice-rings. We have been very successful in eliminating the ice-rings and recover the data underneath. If you are interested you can request the Python script from Michael Chapman at OHSU.

De-icing: recovery of diffraction intensities in the presence of ice rings, Michael S. Chapman and Thayumanasamy Somasundaram

If you need help please e-mail me outside the CCP4BB.

--------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder   

Dr. Thayumanasamy Somasundaram [Soma] Director, X-Ray Crystallography Facility (XRF)			 

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From: James Stroud

I've used a technique called "annealing", which amounts to holding an index card between the cryo stream and the crystal for a few seconds then removing the card quickly.

In my experience, about 70% of the time the diffraction is worse and about 30% of the time the ice rings will be gone with slightly improved diffraction, allowing recovery of a significant range of data. Most of the time, though, I find another crystal that had a better initial freeze, so annealing has never been a life saver--but it could be under dire circumstances.

James

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From: James Holton

Automated outlier rejection in scaling will handle a lot of things, including ice.  Works better with high multiplicity.  Unless, of course, your ice rings are "even", then any integration error due to ice will be the same for all the symmetry mates and the scaling program will be none the wiser.  That said, the integration programs these days tend to have pretty sensible defaults for rejecting spots that have "weird" backgrounds.  Plenty of structures get solved from data that has horrible-looking ice rings using just the defaults.  In fact, I am personally unconvinced that ice rings are a significant problem in and of themselves.  More often, they are simply an indication that something else is wrong, like the crystal warmed up at some point.

  Nevertheless, if you suspect your ice rings are causing a problem, you can try to do something about them.  The "deice" program already mentioned sounds cool, but if you just want to try something quick, excluding the resolution ranges of your ice rings can be done in sftools like this:
select resol > 3.89
select resol < 3.93
absent col F SIGF DANO SIGDANO if col F > 0
and repeat this for each resolution range you want to exclude.  Best to get these ranges from your integration program's graphics display.

In mosflm, you can put "EXCLUDE ICE" on either the "AUTOINDEX" or "RESOLUTION" keywords and have any spots on the canonical hexagonal ice spacings removed automatically.  The problem with excluding resolution ranges, of course, is that your particular "ice rings" may not be where they are supposed to be.  Either due to something physical, like the cooling rate, or something artificial, like an error in the camera parameters.  It is also possible that what you think are "ice rings" are actually "salt rings".  Some salts will precipitate out upon cryo-cooling.  Large ice/salt crystals can also produce a lot of non-Bragg scatter, which means that you can get sharp features far away from the resolution range you expect.  On the other hand, if you have cubic ice instead of hexagonal ice (very common in MX samples), then there are no rings at 3.91A, 3.45A, 2.68A and throwing out these resolution ranges would be a waste. 

Another way to exclude ice is to crank up background-based rejection criteria.  In denzo/HKL2K, you do this with the "reject fraction" keyword, and in mosflm, REJECT MINBG does pretty much the same thing.  There are lots of rejection options in integration programs, and which one works in your particular case depends on what your ice rings look like.  Noone has written a machine-vision type program that can recognize and handle all the cases. You will need to play with these options until the spots you "don't like" turn red in the display.

Of course, the best way to deal with ice rings would be to inspect each and every one of the spots you have near ice rings and decide on its intensity manually.  Then edit the hkl file.


Which brings me to perhaps a more important point: What, exactly, is the "problem" you are having that makes you think the ice rings are to blame?  Can't get an MR solution?  Can't get MAD/SAD phases? 

Ice has a bad rep in MX, and an undeserved one IMHO.  In fact, by controlling either cryoprotectant concentration or cooling rate carefully, you can achieve a mixture of amorphous and cubic ice, and this mixture has a specific volume (density) intermediate between the two.  Many crystals diffract much better when you are able to match the specific volume of the stuff in the solvent channels to the specific volume protein lattice is "trying" to achieve on its own.  A great deal of effort has gone into characterizing this phenomenon (authors: Juers, Weik, Warkentin, Thorne and many others), but I often meet frustrated cryo-screeners who seem to have never heard of any of it!

 In general, the automated "outlier rejection" protocols employed by modern software have taken care of most of the problems ice rings introduce.  For example, difference Pattersons are VERY sensitive to outliers, and all it takes is one bad spot to give you huge ripples that swamp all you peaks, but every heavy-atom finding program I am aware of calculates Pattersons only after fist doing an "outlier rejection" step.  You might also think that ice rings would mess up your preciously subtle anomalous differences, but again, outlier rejection to the rescue. 

Now, that said, depending on automated outlier rejection to save you is of course a questionable policy, but it is an equally bad idea to pretend that it doesn't exist either.  It is funny how in MX we are all ready to grab our torch and pitchfork if we hear of someone manually editing their hkl files to get rid of reflections they "don't like", but as long as "the software" does it, it is okay.  Plausible deniability runs deep.


-James Holton
MAD Scientist

--------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder