Lucas, if your crystals diffract worse at room temp than cryoprotected, it
looks like there may be radiation damage at room temperature, so you may
need to cryoprotect. The change in osmotic pressure may be too much for
your crystals when you're cryoprotecting the crystals by a sudden dunk.
Also, did you just add PEG 400 to the reservoir solution already in the
wells or did you make it afresh with higher precipitant concentration to
compensate for removal into a solution without any soluble protein or
crystalline protein to be in equilibrium with? I always made solutions
afresh unless the crystal was cryoprotected and if it diffracted just fine
by dragging the crystal through a reservoir+cryoprotectant solution.
My crystals did not tolerate sudden changes in osmotic pressure.
Harvest the crystal into 15 uL of reservoir solution with excess
precipitant to compensate for loss of protein to be in equilibrium with (if
I had a crystal from 8% PEG 8000, I used to use 12% PEG8000 with all of the
other components having the same concentration. It was random but I found
one that worked and stuck to it. Sometimes, there may be a solubility
issue, so I had to cut back on a salt but just enough to keep everything
soluble. So I made the solution fresh because my crystals didn't always
appear at exactly the same precipitant concentration. I always set up a
small range of concentrations.
Add 2.5 uL of cryoprotectant (reservoir solution with excess precipitant
concentration AND required concentration of precipitant present in it).
Remove 2.5 uL from a different part of the drop.
Wait 6 min (again, random. 5 min didn't work well, 6 min did).
Repeat with 2.5 uL, 3.75 uL, 5 uL, 7.5 uL. By that time, I achieved enough
cryoprotectant concentration for it to do its job.
I never mixed the drop because my crystals were very fragile and obviously,
I was always looking at it under the microscope to make sure I wasn't
smashing or sucking up my crystal.
Have you tried additive screens? I had tremendous improvement in my
crystals with that. They were very mosaic and overall resolution was only
about 3.7 A or so without additives. With 3% DMSO (from an additive
screen), the size was great, still mosaic, but overall resolution with good
outer shell completeness (I don't remember the exact numbers for data
quality but it was much better than without) improved to about 2.8-ish A.
Good luck!
S.
On Sun, Aug 12, 2012 at 3:31 PM, Yi-Liang (Lucas) Liu
yiliang...@gmail.comwrote:
Hi CCP4ers,
I tested my crystal under room temperature. It still only has low
resolution (7A). Are there any way to improve this? I attached the
diffraction as well. Thanks.
Lucas
On Aug 2, 2012, at 5:27 PM, Roger Rowlett wrote:
Mitegen makes a nice little product that is a plastic tube that will slide
over one of their magnetic cap/loops. If you put some well solution in the
tube and seal the base with apiezon, you can collect quite a bit of data on
the loop mounted crystal before it dries out.
Cheers,
___
Roger S. Rowlett
Gordon Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu yiliang...@gmail.com wrote:
Hi Herman and other CCP3BBers,
Thanks for your suggestions. I didn't see any cracks in the crystal drops
initially. I will certainly try to shot crystals under room temperature and
see what happens. Does the plastic loops fit into the cryo stands Molecular
Dimension sells?
LUcas
On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote:
Hi Lucas,
The funky diffraction pattern is most likely due to a cracked crystal,
resulting in a mixture of slightly differently aligned diffraction
patterns. Were the cracks there before you added the cryprotectant? If
not, the cryoprotectant is definitively to blame. As has mentioned
before, you have to take a shot at room temperature without any
cryoprotectant added, to make sure the bad quality is not due to the
cryoprotectant. Mitegen sells plastic capillaries, which you can slide
over your loop to prevent the crystal from drying out.
Good luck!
Herman
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Yi-Liang Liu
Sent: Thursday, August 02, 2012 4:15 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Enhancing Crystal Quality
Hi,
Thanks for the kindly answers from everyone. I actually haven't tried
different cryoprotectants. I might will give a try next time. I usually
only use mother liquor+30% PEG400. It is noticeable that it has some
patterns (cracks (?)) on the crystal. However, it didn't form icy
rings or etc. The diffraction pattern looks funky too. It looks like it
is twin and the diffraction spot has tails. Does this indicate the
