BIGGER is not always BETTER?
Theoretically it should be better because you have more scattering matter.
If it is
not something has gone wrong in prior steps:
Purification: You were less selective and picked up more heterogeneous
protein.
Crystallization: The bigger crystals grew under conditions that were less
controlled because of changes in the state of protein supersaturation,
precipitant or
temperature. These inhomogeneities contributed to give you bigger, but not
better
crystals.
Cryo-soak: Bigger crystals are more prone to be shocked when transfered to
a
less than optimal cryo-solution. This is a critical step, and crystals do
not like too
much the cryo-chemicals. To test this I tried lower concentrations of
various
cryo-compounds instead of a huge quantity of a single component and in most
cases the mixture was better tollerated than the single components.
Flash-freezing: Bigger crystals will cool more unevenly than small ones. A
change from
liquid nitrogen to liquid ethane could achieve faster cooling because of
the
greater heat capacity of the latter liquid.
Large crystals are more difficult to handle than small ones but after
experimenting with
small ones we can build up a good experimental protocol so that the big
crystals will give exceptionally
good results. I compared small crystals on high intensity beamlines at the
ESRF against
large crystals on BM30 and the big crystals were statistically better.
Unfortunately, it takes
a lot of time and a certain amount of expertize to optimize the
conditions. So ... although I
strongly disagree with Jürgen, I will also advise to start with small ones.
Enrico.
On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen <jubo...@jhsph.edu>
wrote:
Something to add into this discussion is also go fro the tiny crystals
versus the big ones.
BIGGER is not always BETTER - in particular if you try to freeze
directly out of your conditions without an additional cryo-protectant.
And with small or tiny I mean 10 micron, whatever you are capable of
mounting. It is also important to keep the amount of liquid volume
around the crystal low, so rather use a loop in which you scoop the
crystal up instead of having a large loop with lots of liquid.
Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2
leads to a quicker freeze of your material.
If you have the option to anneal your crystal after testing it in the
beam try it out and assess the success or damage, this will be very
different depending on what cryo-additives you have around.
Good luck,
Jürgen
On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote:
One last thing--sometimes crystals can be frozen as is, particularly
if you use mitegen mounts and get nearly all of the mother liquor off
the crystals by dabbing the loop on the dry surface next to the drop
several times. So simple it is always worth a try....
JPK
On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij
<mjvanra...@cnb.csic.es<mailto:mjvanra...@cnb.csic.es>> wrote:
Rationalising it completely may only be possible once you know the
nature of the crystal contacts, i.e. when you have solved the structure.
Until then it is mainly a matter of experimenting.
-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Theresa H. Hsu
Sent: Monday, February 06, 2012 11:00 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Freezing crystal
Hi all
Thanks for all the suggestions which I will try soon.
How do the crystallization condition (PEG vs. salts like ammonium
sulfate) affect the croyprotectant condition? Do factors like presence
of low concentration of high molecular weight PEG (> 2000) mean PEG is
better? Do buffers and salts in protein also important?
Trying to rationalize it :)
Theresa
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu<mailto:j-kell...@northwestern.edu>
*******************************************
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://web.mac.com/bosch_lab/
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
