Phoebe,
In addition to all other comments I like to add that have had several
cases when crystals would not take/like any cryo agent at all. What I
did is added 1% glycerol in the crystallization solution. That did not
change crystallizability of any of the proteins I worked with, but as a
result those crystals would not mind glycerol at all (up to 40% I
tried). One more thing,
the data I get from such grown crystals are better: low mosaicity,
relatively low B-factors. It might be worth trying.
Regards,
Vaheh Oganesyan, Ph.D.
MedImmune, Inc.
Phone: 1-301-398-5851
Facsimile: 1-301-398-8851
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-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]]On Behalf Of
[log in to unmask]
Sent: Thursday, December 06, 2007 11:10 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] SUMMARY: PEG MW vs. cryoprotectivity
Many thanks to all who replied. The answers were remarkably varied -
see below.
My own two bits worth - vitrification of mother liquor doesn't always
lead to a nice, low-mosaicity, ice-free crystal freeze in our hands,
although we're not willing to sacrifice a statistically significant
number of crystals to figure out why. Most of our crystals are
annoyingly fragile, which makes it hard to disentangle the effects of
bad cryoprotection from those of mechanical damage.
Phoebe
From: [log in to unmask]
PEG 4000 has worked for us at high concentration (35-40%)
depending on what else is in there. The same goes for
PEG 3000. You can try increasing the concentration of
the PEG in the reservoir gradually (over the course of many
days) from the % they are grown at up to 25-40%. Hopefully this
will not crack your crystals.
From: Anastassis Perrakis <[log in to unmask]>
in my experience peg 4k reduces the amount of glycerol that you need
but cant act as cryo on its own.
From: Juergen Bosch <[log in to unmask]>
the larger the worse for cryo. But PEG4000 >40% freezes well. PEG8000
needs some addition of smaller PEGs/Glycerol etc.
From: Kevin Jude <[log in to unmask]>
I have used 23% PEG 3350/5% glycerol as a cryoprotectant (JACS 2006 p
3011). The PEG on its own didn't work at that concentration.
It would be enough to test this by making up the solutions and
shooting empty loops, like Elspeth Garman did for glycerol.
From: Ezra Peisach <[log in to unmask]>
I have seen discussions in the past... The easiest thing to do is
test it yourself. Try freezing a small loop of high concentrations of
PEG.
If it forms a clear glass it is worth pursuing...
From: "Jan Abendroth" <[log in to unmask]>
20% PEG 2000 just worked fine, 35% PEG 3350 seems ok too.
also depends on the size of the loop.
From: Edwin Pozharski <[log in to unmask]>
I have used pegmme2000 as cryoprotectant in the past (some 45% of
it), and it worked fine. Indeed, PEG4K s included in Hampton's kit.
From: Buz Barstow <[log in to unmask]>
In our experience with freezing protein crystals under high pressure,
we've found that mid weight PEGs do help a little to enhance the
cryo- protective effect of high pressure, although are not terrifically
effective, especially when at a low concentration of around 5%.
From: "Li Sheng" <[log in to unmask]>
I used 40% w/v PEG 4000 as cryoprotectant.
From: "Moody, Dr P.C.E." <[log in to unmask]>
my experience is that anything over PEG 600 is likely not to be
a reliable cryoprotectant, and 400 is the maximum safe
size.....can't comment on the commercial kits, my cynical nature
would suggest that testing may not be an important part of the
"product pipeline"....Peter
From: Remy Loris <[log in to unmask]>
PEG 4000 is a very good cryoprotectant in the range of 30-35%. If
your crystallization condition includes PEG4000, it is a good idea
for a first trial to find a good cryo condition raise the PEG4000
concentration to 30-35%. Some of the Hampton ctrystal screen
conditions (and other commercial kits as well) that contain PEG4000
do even not need further addition of cryoprotectant (even if in the
corresponding Hampton cryo screen they are diluted with glycerol, one
of the most horible cryoprotectants in common use)
Lower MW PEGS can be useful as well, but the required concentrations
will be higher. Please be aware that in quite a number of cases the
ideal cryo solution can be very far away from the condition in which
the protein was crystallized!
From: "gengxiang zhao" <[log in to unmask]>
Before, I crystallized the complex of a protein with some small
molecules. I use the PEG3350 as a precipatate. When I collect the
dataset using X-ray detector. I only use 23% PEG3350 plus 5% PEG400
as a cryoprotectants. Fortunately, it has been successful.
So, I think that the PEG3350 functions some cryo-protectants.
From: Florian Schmitzberger <[log in to unmask]>
This might not be a direct answer to your question; I have found that
PEG3350 at around 20-25 % (w/v) concentration (JSCG+ screen) - in
combination with ~10 % (v/v) glycerol (which came from the protein
sample buffer) was sufficient to cryoprotect crystals rather well;
without further soaking or handling being necessary.
From: Jennifer Cash <[log in to unmask]>
In our recent experience, larger PEGs work similarly to smaller PEGs
as far as vitrification goes. Additionally, transferring crystals to
a solution of increased PEG concentration (as compared to mother
liquor) can substantially reduce the amount of cryo needed for
freezing and can be more gentle on crystals than just using a high
concentration of cryo. Here is a reference that tests cryoprotective
ability of PEG 2000 & 20000.
J. Appl. Cryst. (2006) 39, 244-251
At 03:55 PM 12/4/2007, [log in to unmask] wrote:
>We've been having a discussion in the lab about whether or not
>middle-sized PEGs such as 4000 can be expected to serve as
>cryoprotectants (and if not, why certain commercial kits are
>formulated the way they are). Can anybody shed some light /
>references on the question of the size of PEGs vs. their ability to
>help in freezing?
> thanks,
> Phoebe
------------------------------------------------------------------------
---------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_A
lphabetically.php?faculty_id=123
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