You've gotten some helpful replies already. I have found the following
reference to be helpful in understanding some of the physics behind
damage incurred during the crystal cooling process and a general
strategy to help avoid it. It expands upon what's already been said -
that larger crystals are more prone to distress during cooling. This
and other papers from the same group contain useful information and advice.
A General Method for Hyperquenching Protein Crystals
Matthew Warkentin and Robert E. Thorne
Struct Funct Genomics. 2007 December ; 8(4): 141–144.
doi:10.1007/s10969-007-9029-0.
Best,
-Andy
On 4/15/2010 4:48 AM, Mark J. van Raaij wrote:
and don't forget to check diffraction without freezing.
Mark
On 15 April 2010 10:37, Anastassis Perrakis <a.perra...@nki.nl
<mailto:a.perra...@nki.nl>> wrote:
Hi -
My two cents:
First, you say:
I assume the bigger crystal might have lot of solvent which
prevent for high resolution. If it is true what could be the
best way to dehydrate crystal without affecting crystal quality?
I think this assumption is confusing. If the crystals were grown in
the same drop/condition, they have identical percentage solvent
content. Thus, you do not want to look at dehydration, the
'percentage solvent content' is fine. What you want to look at is
the mechanics of vitrification. Big crystals, are simply hard to
freeze: because of their volume they cannot be vitrified as rapidly
and uniformly as smaller crystals. I will not be surprised if there
are papers that quantify that, but what I am saying here is only
from experience and adding a 'logical' explanation to that experience.
Thus, I would simply stay with the smaller crystals (I have a
feeling that you 'small' crystals are 'big' for many other people)
and be happy they diffract to 2.5 A (is that SR or RA?)
A.
On Apr 15, 2010, at 3:16, syed ibrahim wrote:
Dear Jurgen and Ho Leung
To add few more point regarding my question:
1. Crystal was first frozen in LN2 and then transfered to cryo
stream (in presence of LN2 in vial)
2. Anealing did not help (both short time and long time) -
perhaps the crystal dies.
3. Spots are clear to available resolution (is: 6-7A). In the
high resolution region there is no spot but looks like smear in
the whole area.
4. The crystal was approximately 1.0mm length and 0.4mm dia. I
mounted on 0.5mm loop. So the liquid around the crystal was very
less. I deliberately avoided more solvent in the loop to help
diffraction.
Thanks
Syed
--- On *Thu, 4/15/10, Jürgen Bosch /<jubo...@jhsph.edu
<mailto:jubo...@jhsph.edu>>/* wrote:
From: Jürgen Bosch <jubo...@jhsph.edu <mailto:jubo...@jhsph.edu>>
Subject: Re: [ccp4bb] Cryo Vs crystal size
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Date: Thursday, April 15, 2010, 3:46 AM
There are a couple of additional factors not taken into
account here.
1. LN2 versus frozen in strem or propane etc
2. did you try to flash anneal the larger crystal
3. smeary diffraction from the big crystal or not ?
4. how much residual solvent was around your crystal when
freezing ?
In general smaller crystals are anyhow better in my hands.
Jürgen
On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:
Hi All
I had two crystals grown in same well, one is small and other
is 10 times bigger. I treated both crystal in same cryo and
same time. The smaller one diffracted to 2.5A and the bigger
one to 6-7A. I was expecting the bigger one to diffract high
resolution.
I assume the bigger crystal might have lot of solvent which
prevent for high resolution. If it is true what could be the
best way to dehydrate crystal without affecting crystal quality?
Thank you
Syed
PS: Taken care of less solvent to be present in the loop
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/>
*P** **please don't print this e-mail unless you really need to*
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
--
Mark J van Raaij
http://webspersoais.usc.es/mark.vanraaij
http://www.ibmb.csic.es
