Chris,
As others have said, using sugars as cryoprotectants is a good first
choice. However, we have run into problems freezing crystals with
sugars when the primarily crystallization reagent is a salt at high
concentrations (0.8-2M). Although 0.4M ammonium phosphate, is not
particularly high, you might try ammonium formate or sodium malonate,
sometimes even sodium citrate works (1.0-1.2 M). My worry about any
crystals grown in phosphate is that the phosphate anion may be crucial
to crystals growth, and its displacement by like ions (sulfate) may be
detrimental. If it is not in your case, you might also try lithium
sulfate. We have used mixtures of sodium citrate and lithium sulfate
to freeze crystals grown in 0.8 M sodium citrate.
One other point is that sometimes the crystals need to have a bit of
the cryoprotectant as a component of crystallization. I have seen
cases where crystals could not be transferred into glycerol, but
adding 1-3% glycerol to the crystallization mix yielded crystals that
could be transferred into glycerol for freezing.
You have a lot more options to try, but a drop on a coverslip may not
be the best way to test freezing. Proper freezing depends on having
maximal heat transfer, sometimes that can be defeated by having
"large" heat reservoirs (i.e., big drops and the big coverslip) and
insulators (i.e., glass coverslips). Freezing works because the
objects size (drop and crystal) is small enough to allow rapid and
relatively isotropic heat transfer. We alway use a slightly larger
loop to test our cryoprotectants.
When using ammonium phosphate, watch out for the formation of struvite
(NH4MgPO4), a type of kidney stone. They are lovely looking (often
octahedral) crystals that easily grow in ammonium phosphate; although
magnesium phosphate can be soluble up to ~12 mM, the presence of
ammonium markedly increases the formation of struvite, even with
micromolar (contaminating) concentrations of magnesium.
Cheers,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: rmgarav...@gmail.com
****************************************************************
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
Of Chris
Ulens
Sent: Thursday, May 26, 2011 5:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen
phosphate cryo
Hi,
I would like to get recommendations for a proper cryo solution for a
crystallization hit from the Hampton crystal screen Ammonium di-
hydrogen
phosphate 0.4M. I tried increasing glycerol up to 30% with the same
ammonium
phosphate concentration or increasing glycerol up to 30% in the
presence of
1.3M ammonium phosphate. Both gave iced up drops (I only tried quick
and
dirty tests by dipping a cover glass in liquid nitrogen).
Thank you.
-Chris
