Hi Mary,
There is another expt. you might look into after trying out the different
cryoprotectants, small vs. large crystals, capillary mounts, etc. as suggested.
Dr. Robert Thorne's group (from Mitegen & Cornell U.)) has been doing some
studies on flash freezing procedures and effect on mosaicity/diffraction
quality. Some of the conclusions and recommendations from these experiments
deals with the effect of cooling rate during flash freezing (by dunking in
liquid nitrogen) and the effect on mosaicity.
If I remember correctly, they found that crystals they tested had much
lower mosaicity during diffraction data collection if instead of dunking the
looped-out crystals directly in the container with liquid nitrogen, they dunk
it in the container which has dry liquid nitrogen blowing laterally along the
surface. This changes the environment at the liquid/air interface near the
container allowing for much faster cooling rates than otherwise, resulting in
an improved diffraction quality and reduced mosaicity.
I haven't tried it myself but the results seemed convincing. I know that
sector 8 beamlines at the ALS have a modified apparatus to do this (look for
Corie Ralston). You can get more information from them or contact Robert Thorne
directly.
BTW, ALS beamline 12.3.1 has the FMS system if you want to try that out.
Regards,
Debanu.
---
Debanu Das,
Stanford Synchrotron Research Laboratory,
SLAC, Menlo Park, California.
________________________________
From: CCP4 bulletin board on behalf of Kris Tesh
Sent: Wed 7/11/2007 8:35 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Help with reducing crystal mosaicity
At the risk of sounding too commercial, here are some suggestions:
1. See if you can either place your crystallization tray in a cold room or
crystallize it there. Mount your crystal in a capillary here so that at
whatever lower temperature you use, the distillation of solvent will be
reduced.
2. If capillaries are too hard to work with, even a quick diffraction image
out of a loop, while the crystal is drying out, may give you some
information. Alternately, I have saturated my stock solution with sugar
from the coffee room and swished my crystal through this for getting a quick
diffraction image. In a rare case, the crystal actually diffracted for
several hours at room temperature.
3. Someone mentioned using Paratone oil. This is good, but a little thick
and sticky. So, I use PerFluoroPolyEther (PFPE) as it is much less viscous
and is colorless. I buy FOMBLIN Y VAC 14/6 from SigmaAldrich (Cat #
317934-100G). PFPE is mildly hydroscopic, so it is best to open the bottle
infrequently (I withdraw ten 0.5 ml aliquots whenever I open the bottle).
4. The Free Mounting System (FMS) is ideal for RT screening. So long as
you do not have a highly volatile component in solution, then you can use
the standard FMS at temperatures in the range of 15 to 25 C. If you do have
a volatile component, then Proteros bistructures GmbH (www.proteros.com) has
the only FMS with the capability of adding solvents to the gas stream...and
with some compensation, they will gladly help you with this.
Kris
------------
Kris F. Tesh, Ph D
Director, Macromolecular Products
Rigaku Americas Corporation
9009 New Trails Drive
The Woodlands, TX 77381 USA
001 281 362 2300 x 144
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Benini, Stefano
Sent: Wednesday, July 11, 2007 7:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Help with reducing crystal mosaicity
Dear Mary,
the problem encountered with cryoprotectans is the change in the solution
surrounding your crystal as they may not be present in your crystallisation
conditions or you need to increase their concentration to make them act as
cryoprotectant.
So I don't thik is the cryoprotectant itself as it is you mother liquor..
test it in a capillary so you could rule out that freezing is the problem
of course you don't want to find new crystallisation conditions when you
tried everything and I know what it means!!
I would try to improve crystal quality using additives or ligand etc....,
your long axis is very worrying as well!!! may be some magic additive could
change the packing and givew better diffraction too
I also had crystals growing from MPD, unfortunately there was a
non-cleavable his-tag
so I could not hope that metals would help..... and other additives did not
make any difference
then my contract finished and so did the project!
I hope you still have a lot of time available to try different things but I
would not waste time trying to change your cryo
ciao
Stefano
Stefano Benini PhD
AstraZeneca UK
Structural Biology
Mereside 50S38
Alderley Park
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
Patrick Shaw Stewart
Sent: 11 July 2007 12:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Help with reducing crystal mosaicity
Just a thought, Mary - going back to your original question about MPD. I
extracted the crystallization conditions from REMARK 280 of 3939 PDB entries
a couple of years ago. The average concentration of the MPD used was high -
38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You
can see the data at www.douglas.co.uk/top14.htm
I thought this information could be useful if you want to replace some of
the MPD with another precipitant (or cryoprotectant).
Best wishes
Patrick Shaw Stewart
--
[EMAIL PROTECTED] Douglas Instruments Ltd.
DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK
Directors: Peter Baldock, Patrick Shaw Stewart, James Smith
http://douglas.co.uk <http://douglas.co.uk/> or
http://www.douglasinstruments.com <http://www.douglasinstruments.com/>
Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034
Regd. England 2177994, VAT Reg. GB 480 7371 36
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mary
> Fitzgerald
> Sent: 09 July 2007 23:05
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Help with reducing crystal mosaicity
>
> Help please!
>
> I'm looking for some new ideas. I have crystals that come out of a
> sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium
> acetate and MPD for the well solution. The MPD concentration is
> sufficient to act as a cryoprotectant. Currently, I directly freeze
> these crystals in liquid nitrogen. When I collect data, I typically
> have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is
> further complicated with a weakly diffracting crystal (4-5 A) that has
> a long unit cell axis of ~500 and often twinning.
>
> It has been suggested to me that the cryoprotectent is a problem. I
> haven't checked the diffraction at room temperature, yet. Please no
> suggestions of finding a different crystal form as that's not a
> consideration at the moment. I have my reasons. I did find one
> crystal that has lower mosaicity (0.5 to 0.8) but had weaker
> diffraction then the typical crystal. Attempts at flash cryoannealing
> have not helped.
>
> So, what's a good way to change the cryoprotectant if the
> cryoprotectant is the precipitant? I've considered trying dehydration
> but wasn't certain if that would help with the mosaicity.
>
> Thanks for any ideas,
>
> Mary X. Fitzgerald
> Postdoctoral Associate
