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 <[log in to unmask]>
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
> <[log in to unmask]<mailto:[log in to unmask]>> 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:[log in to unmask]] On Behalf Of
> Theresa H. Hsu
> Sent: Monday, February 06, 2012 11:00 PM
> To: [log in to unmask]<mailto:[log in to unmask]>
> 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: [log in to unmask]<mailto:[log in to unmask]>
> *******************************************
>
> ......................
> 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: [log in to unmask] Fax: 33 (0)1 69 08 90 71
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