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Hi Zhiyi,

There are two ways you can go about this (RT data collection). Either mount the crystals in capillaries (not all beam lines at synchrotrons have sufficient space on the goniometer setup to allow data collection on capillary-mounted crystals) or use a Humidifier (the latter is briefly discussed, for example, on web page http://www.esrf.eu/UsersAndScience/Experiments/MX/Cryobench/Instruments/microspec ).

If nothing is done, the crystals will rapidly dry up and "die".

Fred.

  

Message du 27/01/10 08:53
De : "Zhiyi Wei" 
A : [log in to unmask]
Copie à : 
Objet : Re: [ccp4bb] Crystal rescue


Thanks for so many quick responses!

Actually, I have test several different cryo-protectants, including
glycerol, EG, and PEG400. I did not see much differences between these
cryo conditions. So, I choose glycerol.

I would like to test my crystals in RT. But I don't know how to do
this. Just mount crystal to the X-ray machine without cryo stream? Or
I should use capillaries?

Zhiyi

On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry  wrote:
    

Tascimate can be used as the cryo as well. I have had experience with
crystals in similar condition and moved the crystals to a 20%
increased Tascimate solution and they froze well.

I agree with Ezra, room temperature mount your crystal before
freezing. It is the only way to know the true problem.


Kelly
*******************************************************
Kelly Daughtry
PhD Candidate
Department of Physiology and Biophysics
Boston University School of Medicine
590 Commonwealth Ave
R 390
Boston MA, 02215
(P) 617-358-5548
*******************************************************



On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter
 wrote:
      

Dear Zhiyi,


Ezra is exactly right, of course. The Oxford Diffraction PX Scanner
system can assess the diffraction qualities of (putative) protein
crystals in situ - in the crystallisation plate. So, directly, you
would discover if your 'big and beautiful' crystals actually diffract
well... in their mother liquor under ambient conditions and before the
addition of any cryo-protect. Do you have a friend or neighbour with
a PX Scanner ? If not, please feel most welcome to contact
Oxford Diffraction: we would be pleased to assist if at all possible.


Good Luck and Best Wishes,

Marcus Winter.

www.oxford-diffraction.com




-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Ezra Peisach
Sent: 26 January 2010 16:01
To: [log in to unmask]
Subject: Re: [ccp4bb] Crystal rescue

First you need to establish if it is your cryo conditions or the
crystals. Depending where you are - they might have the equipment to do

a wet mount - without freezing. Yes the crystal will not last - but
then you know if the problem is in the
crystal. If it is - you need better crystals. If it is the cryo - you
need to work on that. Tacsimate is mixture of alot of different
compounds - but the smears are too close together to be a small salt
crystal on top...

Good luck,

Ezra

On 1/26/2010 10:42 AM, Zhiyi Wei wrote:
        

Dear all,

I got a problem with my crystals. I have two total different proteins
that both can be crystallized in the condition with PEG3350 and
          

Tacsimate
        

(although the concentrations are different) with different shapes. The
crystals look big and beautiful. However, when I test them in
          

synchrotron,
        

both of these two types of crystals showed poor diffractions. As
          

showed in
        

the attached diffraction image, the diffraction is up to ~4 A but
          

smear in
        

one direction while<8 A in the other direction. The interesting thing
          

is
        

that the diffraction pattern is similar for all crystals (from two
          

different
        

proteins) that I tested without exception although they belong to
          

different
        

space groups. So, I wonder whether these kind of pattern is caused by
Tacsimate (I don't know what it is) and how to rescue these crystals.
          

Any
        

suggestions or comments?

Thanks a lot!

Best,
Zhiyi

          

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Matthew Bowler
Structural Biology Group
European Synchrotron Radiation Facility
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FRANCE
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