[ccp4bb] On Behalf Of Katherine Sippel, Re: [ccp4bb] High Salt Cryo

2014-02-24 Thread Kiefersauer, Reiner
Hi,

what was not mentioned in the ccp4 discussion is

The use of trimethylamine N-oxide as a primary
precipitating agent and related methylamine
osmolytes as cryoprotective agents for
macromolecular crystallography

Acta Cryst. (2012). D68, 69-81

I found it as a very potent compound for cryo-preservation of crystals.

Kind regards,
Reiner

--
Dr. Reiner Kiefersauer
research scientist

Proteros Biostructures GmbH
Bunsenstraße 7a
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Germany

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Re: [ccp4bb] High Salt Cryo

2014-02-21 Thread Katherine Sippel
-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.comhttps://
 urldefense.proofpoint.com/v1/url?u=http://archive.comk=
 7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4
 V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%
 2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3d
 d6e2ded91923c614e670857e10916c687e [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.comamp;k=
 7DHVT22D9IhC0F3WohFMBA%3D%3D%0Aamp;r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4
 V0aIwH4RJhyZU%3D%0Aamp;m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%
 2FzyGkSwsbIoq92CSnOk%3D%0Aamp;s=3cfbf18821b5b59934971bf583cf3d
 d6e2ded91923c614e670857e10916c687ehttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=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 9449Lab
 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=enuser=Kvm06WIoPAsCpagesize=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




-- 
Nil illegitimo carborundum* - *Didactylos


Re: [ccp4bb] High Salt Cryo

2014-02-21 Thread Keller, Jacob
If you need phases, you might change the salt ion(s) to something with 
significant anomalous signal, i.e., Rb+, Cs+, Br-, I- instead of Na+ and Cl-. 
With such high ion concentrations, you should get some really high-occupancy 
sites. In any case it is sometimes handy to have experimental phases if things 
don’t go the way you thought with MR.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katherine 
Sippel
Sent: Friday, February 21, 2014 11:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] High Salt Cryo

I want to start off by thanking everyone. The replies, both on- and off-board, 
were speedy and numerous. I apologize for the delay in expressing my gratitude; 
I was implementing your wonderful suggestions. I have put together a summary 
for the archive.

To recap, I was looking for suggestions to cryoprotect crystals from 3 M NaCl 
crystallization conditions excluding ethylene glycol or PEG completely and 
avoiding glycerol and sucrose due to apparent crystal instability.

-Several people confirmed that 4 M NaCl should be sufficient.

-There were several suggestions for cryosalts with malonate and formate being 
the most frequent. Lithium salts were also put on the table including partial 
or complete substitution of NaCl with LiCl, both as a soak or a crystallization 
solution.

-As an end run around the apparent glycerol instability, stepwise transfers and 
quick dips with glycerol were suggested. Glycerol supplemented with xylitol was 
also thrown into the mix.

-There was one tale of sucrose being successfully added into a crystallization 
condition when direct soaks were unsuccessful

-There were several confirmations regarding the success of Paratone and 
Paraffin oil with pro-tip of dehydrating the paraffin oil in a speed-vac 
overnight.

-Additional suggestions included 50-75% saturated sugars and 6.5 M proline.

Since I’m a good scientist I will include the reference section. I found it 
very useful.

-Cryosalts: suppression of ice formation in macromolecular crystallography. K. 
A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001

-Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown 
macromolecular crystals. T. Holyoak et. al.  Acta Cryst. (2003). D59, 2356-2358

-Proline: Mother Nature's cryoprotectant applied to protein crystallography. 
T.A. Pemberton et. al. Acta Cryst. (2012) D68, 1010-8.

-Effects of cryoprotectant concentration and cooling rate on vitrification of 
aqueous solutions. V. Berejnov et.alhttp://et.al. J. Appl. Cryst. (2006) 39, 
244-251

-A comparison of salts for the crystallization of macromolecules. A. McPherson. 
Protein Sci. (2001) 10, 418-22

-Strategies for protein cryocrystallography. L. Vera, E. A. Stura. Crystal 
Growth  Design (2013) 14(2), 427-435

Thank you all again. I really appreciate your time and energy.

Cheers,
Katherine



On Wed, Feb 19, 2014 at 9:23 AM, Enrico Stura 
est...@cea.frmailto:est...@cea.fr wrote:
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.plmailto: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

Re: [ccp4bb] High Salt Cryo

2014-02-19 Thread Mooers, Blaine H.M. (HSC)
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-

X-ray lab webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory

Small Angle Scattering webpage: 
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0

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.comhttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e
 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


Re: [ccp4bb] High Salt Cryo

2014-02-19 Thread Karolina Michalska
 

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.comhttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=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.comamp;k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Aamp;r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Aamp;m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0Aamp;s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e

Re: [ccp4bb] High Salt Cryo

2014-02-19 Thread Enrico Stura

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.comhttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=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.comamp;k

[ccp4bb] High Salt Cryo

2014-02-18 Thread Katherine Sippel
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 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


Re: [ccp4bb] High Salt Cryo

2014-02-18 Thread Roger Rowlett
How about a short swish in well solution + 25-30% glucose? Doesn't take 
long to cryoprotect, just a quick sufrace coat. Sodium malonate? We just 
froze some really fragile crystals from 1.8 M sodium formate in 3 M 
sodium malonate and they held up really well. (Still didn't improve 
their diffraction, though--but at least they did not crack or disintegrate.)


___
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 2/18/2014 1:08 PM, Katherine Sippel wrote:

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 http://archive.com 
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