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
> <[log in to unmask]> 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: [log in to unmask]
>>
>> 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 [[log in to unmask]] On Behalf Of
>> Katherine Sippel [[log in to unmask]]
>> Sent: Tuesday, February 18, 2014 12:08 PM
>> To: [log in to unmask]
>> 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: [log in to unmask] Fax: 33 (0)1 69 08 90 71
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