Methods for dehydrating crystals

From: Andrea L Edwards
Date: 26 August 2011 21:53
Hi all,

What are the most successful methods you know of for dehydrating a crystal prior to freezing it? I am trying to push the resolution of my crystals.

Thanks,
Andrea
----------

From: Edward A. Berry

First of all, be aware that not all crystals are improved by
dehydration. Some need to be drier, some need to be wetter.
If you have long or large crystals that extend outside
the loop, you can get a clue by comparing diffraction at the
outside tip (driest) with the center of the loop (wettest).
We've seen dramatic differences in both directions, and
the ones that diffract best outside (beef orthorhombic
cyt bc1) were greatly improved by dehydration, while for
the ones that diffract best in the center (chicken complex II)
we got the best diffraction when they were so wet we started
to get ice rings. Dehydration destroyed them.

Our best dehydration results were with simply holding the
crystal in the air 30 sec to 2 min after mounting before
plunging into LN2. Also something about pushing the crystal
to the edge of the drop where the peg is getting sticky
before fishing. be aware the result may depend on relative
humidity and temperature.

My grand plan for optimizing
dehydration (if the FMS is not available) was to mount
crystals on cryocap pins and screw the caps into vials
containing 0.5 ml of different concentrations of glycerol.
(Pin being short enough that the crystals are suspended
in the air over the solution)
Wait for a few hours at RT or 4* then unscrew the cap and
rapidly freeze. Never really got good results though.
And if the humectant is really wet, the crystal soaks
up so much water it falls out of the loop into the
humectant!

Ed

----------
From: Savvas Savvides

Dear Andrea

check out:

Post-crystallization treatments for improving diffraction quality of protein crystals.

Heras B, Martin JL.

Acta Crystallogr D Biol Crystallogr. 2005 Sep;61(Pt 9):1173-80.

All the best

Savvas

----------
From: Matthew BOWLER
Dear Andrea,
   as others have suggested there are many ways to dehydrate crystals by increasing the concentration of salt/precipitant or adding glycerol/EG/PEG 200-400 to the solutions surrounding your crystals.  I have always found controlled dehydration using a specific device  to be much better.  The most common are the FMS and the HC1, the great advantage is that any changes that are induced by dehydration can been observed immediately by diffraction and the whole process can be thoroughly characterised.  You never know if it is going to work but it is always worth a try, good indications are relatively large solvent content/solvent channels or that you have already observed variation in unit cell parameters after cryocooling.  In Europe the HC1 is available at the ESRF (http://go.esrf.eu/HC1b - you can apply for rolling access here, http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal), Diamond, MAXLab and I think BESSY.  I believe the FMS is available to use at Proteros.  Here are some links to papers describing the use of these devices, good luck!  Matt.


FMS - http://journals.iucr.org/j/issues/2000/05/00/he0257/index.html
HC1 - http://scripts.iucr.org/cgi-bin/paper?S0907444909037822
and http://www.sciencedirect.com/science/article/pii/S1047847711000499

--
Matthew Bowler
Structural Biology Group
European Synchrotron Radiation Facility
B.P. 220, 6 rue Jules Horowitz
F-38043 GRENOBLE CEDEX
FRANCE
===================================================

----------
From: Hargreaves, David
Hi Andrea,

If you haven't already done so it might be worth trying a room temperature mount (capillary or in-situ) to get a feeling for how well the crystals diffract to start with.

Dave

David Hargreaves
Associate Principal Scientist
_____________________________________________________________________
AstraZeneca