Dear all,



From Leonid's reply earlier you can see a problem some of us have been having for a while now, when looking for literature regarding dehydration. Most of you that perform dehydration either don't consider it happening or don't report it in great detail in your publications. This is only understandable because it isn't the focus of your work and it only helps you get to where you want to get to.



I'm trying to get an up to date picture of what is out there but I haven't got the time or eyes to go through everyone's methods to pick the couple of lines that describe your particular method. I really want to find out what is being done to be able to give people better advice.



So: Could people out there that think that in their particular projects dehydration/hydration had an effect send me a ref. or a short description? (can be done outside the BB to not spam everyone) I will duly acknowledge everyone!!



By dehydration I mean:



1 Soaking with increasing concentration of precipitants or salts

2 By equilibrating against a new precipitant or salt (by vapour diffusion or dialysis)

3 By letting the drops dry (controlled or uncontrolled)

4 by using an FMS/HC1/MicroRT or any other gadget

5 By some other magical trick you may have



Thank you all for your help,



Regards



Juan



====================================

Juan Sanchez-Weatherby, PhD

Beamline Scientist - I02

Macromolecular Crystallography Group

 

Diamond Light Source Ltd

Diamond House DR1.64

Harwell Science and Innovation Campus

RAL, Chilton, Didcot

Oxfordshire

OX11 0DE

United Kingdom

 

Tel: +44 (0)1235 778661

Mob:+44 (0)7795 641259

Fax:+44 (0)1235 778052

 

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http://www.diamond.ac.uk

==================================== 



-----Original Message-----

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Leonid Sazanov

Sent: 15 January 2013 19:32

To: ccp4bb

Subject: Re: [ccp4bb] crystal dehydration



In case if dehydration needs to be done slowly and under tight control of all parameters, one possibility is to use micro-dialysis  buttons.



We used it for a large membrane protein complex and diffraction improved from ~7 to 2.7 A. The crystal is fished out and put into mother liquor solution in the button, sealed with dialysis membrane and the button is then placed into about 5 mls of mother liquor with slightly higher PEG concentration. Then you just exchange outside buffer every day or so for solutions containing higher concentrations of PEG. We went from ~9 to 30 % PEG4000 in about a week. You can easily observe crystal under microscope and if it cracks - you went too far/too quickly with PEG and need to use a bit less next time. Also, this method allows you to control all other components of the dehydrating solution - we needed to decrease salt concentration at the same time as increasing PEG. You can also introduce/increase cryo-protectant concentration at the same time. With these crystals, otherwise excellent dehydration machines already mentioned did not work, possibly because the process had to be really slow. The reference is here: http://www.ncbi.nlm.nih.gov/pubmed/21822288



Best wishes.