Dear All,
I would like to point out that the conditions 1.8 - 2.0 M NaCl are not
considered High Salt as
NaCl is soluble to 5M and a 2X solution (i.e. 4M NaCl) is possible. Also
NaCl contrary to
ammonium sulfate, citrate, phosphate, etc. is compatible with polyethylene
glycol without phase
separation problems.
This means that with 1.8 - 2.0 M NaCl you have an vast repertoire of
possible ways
to cryo-protect crystals and with the vast repertoire you gain a good
possibility of finding
conditions that enhance diffraction:
see:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.
Crystal Growth & Design,
http://pubs.acs.org/doi/full/10.1021/cg301531f
For me the definition for "High Salt" is that 2X for the precipitant
component is not possible.
Enrico.
On Wed, 19 Feb 2014 16:06:27 +0100, Karolina Michalska
<dzi...@amu.edu.pl> wrote:
4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M
NaCl.
Karolina
W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a):
For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M
AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4
uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds
and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4
M. We do not bother withdrawing aliquots to maintain a fixed volume.
You may need to tweak the volumes to optimize the resulting
diffraction. You can also break the additions at given concentration
into smaller aliquots to reduce the osmotic shock. This approach is
much gentler than transferring the crystal directly to 3 M sodium
malonate. Do not leave the drop exposed to the air for more than 3
minutes or so because salt crystals will start to grow. When there are
multiple crystals in a drop, often the unused crystals in the very high
salt solution will still diffract well up to a year later if the
crystallization chamber is resealed well; their diffraction might even
improve with the prolonged exposure
to high salt.
Blaine Mooers
Assistant Professor
Department of Biochemistry and Molecular Biology
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center Rm. 466
Shipping address:
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419
Letter address:
P.O. Box 26901, BRC 466
Oklahoma City, OK 73190
office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910
e-mail: blaine-moo...@ouhsc.edu
Faculty webpage:
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
[1]
X-ray lab webpage:
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
[2]
Small Angle Scattering webpage:
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
[3]
________________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Katherine Sippel [katherine.sip...@gmail.com]
Sent: Tuesday, February 18, 2014 12:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High Salt Cryo
Hi all,
I'm looking for a cryo condition for high NaCl (3+ M) crystallization
condition. I would do it the proper way, but our beam/cryostream is
down.
I've tried a bunch of things at the moment. Ethylene glycol and PEG 400
nuke the crystals immediately even at low concentrations. Prolonged
exposure to glycerol and sucrose starts to break them down so I'm
thinking that the diffraction will probably suffer. I can't find any
reports of NaCl's viability as a cryosalt. I've got Paratone/Paraffin
oil/Mitegen's LV cryo oil on tap but I was hoping to not put all my
eggs in one basket.
I tried the ISRDB database through
archive.com<https://urldefense.proofpoint.com/v1/url?u=http://archive.com&k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0A&r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e
[4]> without any luck (no search function). I've gone to the PDB
searching for similar crystallization conditions and looked up the
papers for their cryos, but they are all glycerol. Google gives me the
same.
I thought I'd see if anyone on the bb has an anecdotal "this worked for
us" story. I would love to hear it.
Thank you for your time,
Katherine
--
"Nil illegitimo carborundum" - Didactylos
Links:
------
[1]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
[2]
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
[3]
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
[4]
https://urldefense.proofpoint.com/v1/url?u=http://archive.com&k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0A&r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e
--
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
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
