Re: [ccp4bb]: New xtal handling method?

["Tom Caradoc-Davies" <toms_postingacct@hotmail.com>]

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Hi all,
        thanks for the Emails! I should have been a bit more specific. 
Thanks for the info that that oil has been used for xtal mounting over the 
years (esp Paratone) and it's use is common in small molecular xtallography.

What I was wondering about is do people routinely use oil to protect xtals 
(produced by normal vapour diffusion expts) on loops from drying during 
transit from cryo-solution to cryo-stream?
I'm sure we are all familiar with the "get the loop containing the xtal from 
the cryosolution to the cryostream before it dries out" game and have seen 
xtals lost/damaged in the rush (well in my hands anyway!).

Using a thin film of parrafin oil a xtal can be looped out of cryo-solution 
and is protected for at least 10 mins. So instead of the rush you could have 
as much time as you want to move from the microscope to the goniometer and 
get the xtal aligned in the cryo-stream.

I tested this by exposing an xtal to X-rays while it was in a loop covered 
by a film of oil (no capillary or cryo-stream to protect it) and this 
protected it from dehydration for over 45 minutes.

Micro-bridges filled with 40microL of paraffin oil and containing a 6microL 
drop of cryo-solution full of nice xtals were left uncovered for a week on a 
microscope stage and after that time xtals still diffracted as well as 
controls. This is a real help for me as I can screen a range of xtals from 
different expts without having to open and close vapour diffusion expts or 
rush because 1microL drops are drying out while I'm trying to loop out the 
best xtal.

I am suggesting combining the use of a cryo-solution with oil rather than 
the technique where the xtal is placed in oil and a wick is used to remove 
mother liquor from the xtal. Oil is layered over the drop from the vapour 
diffusion experiment and then the xtal looped into a drop of cryo-solution 
under 40microL of paraffin oil in a micro-bridge. If you have to move a xtal 
through a series of cryoprotectant concentrations, placing paraffin oil over 
the sitting drops allows the xtal to be protected from drying.

If you look at the loop under a microscope the xtal has a thin film of 
cryo-solution coating the planes of the xtal and this is held in the oil 
film. Using this technique there is a lower diffuse scatter on X-ray images 
than using normal xtal handling methods and I assume that this is due to 
less volume of cryo-solution on the xtal than a normal drop and the paraffin 
oil not producing much diffuse scatter but this needs more research. Oh and 
I use a slightly larger loop than normal to get a thin film of oil and less 
cryo-solution on the xtal.

I guess I just found this trick to be so incredibly useful that I wondered 
if it could be of use to anyone else?
Again if this is old hat I apologise and sorry about the length of this 
post!
Cheers,
       Tom


        Tom Caradoc-Davies
        Biochem Dept
        Univ. Otago
        New Zealand



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