Re: [ccp4bb] Crystal rescue

[Ezra Peisach]

MiTeGen (www.mitegen.com) sells a RT device  - plastic capillary to put 
over loop... I do not know how well it would work for a long data 
collection - but people here have used it to evaluate their crystals....

Ezra

On 1/27/2010 3:58 AM, Flip Hoedemaeker wrote:
Zhiyi,

You can use a thin cap over your cryo loop, just put a drop of mother 
liquor in the top, place over the loop and make it airtight at the 
base.  Not sure who sells these things though, I guess you can make it 
from a capillary too. Then remove the cryo stream or put it at a temp 
above freezing, say 253K.

Flip

Zhiyi Wei wrote:
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 <kddau...@bu.edu> 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
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On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter
<marcus.win...@oxford-diffraction.com> 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:ccp...@jiscmail.ac.uk] On Behalf Of
Ezra Peisach
Sent: 26 January 2010 16:01
To: CCP4BB@JISCMAIL.AC.UK
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