Re: [ccp4bb] suggestions for cryoprotectant

[Walter Novak]

Hi Firdous,

For salt conditions try malonate:
Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2356-8. Epub 2003 Nov 
27.
Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown 
macromolecular crystals.
Holyoak T1, Fenn TD, Wilson MA, Moulin AG, Ringe D, Petsko GA.

Best,
Wally
-- 
Walter R.P. Novak
Associate Professor and Chair of Chemistry
Wabash College
301 W. Wabash Ave.
Crawfordsville, IN 47933
Phone: 765-361-6407
Fax: 765-361-6149

Date:Tue, 23 Oct 2018 13:29:10 -0700
From:Kevin Jin 
Subject: Re: suggestions for cryoprotectant

Dear Firdous,

Probably other people already suggested above, and I don't know which kind
of protein in your case.  Here is my suggestion, you may try sugar solution
or polysaccharide solution as cryoprotectant. The whole idea is to provide
alternative H-bond network to support protein freezing. Indeed, some
startups are using similar ideas to develop cryoprotectants for medical
applications.

Best,

Kevin



On Fri, Oct 19, 2018 at 2:58 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I 
also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization 
hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

 





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Re: [ccp4bb] suggestions for cryoprotectant

[Kevin Jin]

Dear Firdous,

Probably other people already suggested above, and I don't know which kind
of protein in your case.  Here is my suggestion, you may try sugar solution
or polysaccharide solution as cryoprotectant. The whole idea is to provide
alternative H-bond network to support protein freezing. Indeed, some
startups are using similar ideas to develop cryoprotectants for medical
applications.

Best,

Kevin



On Fri, Oct 19, 2018 at 2:58 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/



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Re: [ccp4bb] suggestions for cryoprotectant

[Magnus Claesson]

Hi Firdous,

Dry mineral oil usually works well, and it does not alter the 
crystallisation drop composition. Even better if you are using sitting 
drop. Just add enough mineral oil to cover the crystallisation drop, and 
you even have a lot more time to pull your crystal out before the high 
salt condition crystallises around the protein crystals. Eventual ligand 
could also be added to the crystallization drop before pulling the 
crystal through the mineral oil.


Cheers,
Magnus



Den 19/10/18 kl. 23:57, skrev Firdous Tarique:

Dear members

I have got beautiful crystal hits in SaltRx screens which are not 
diffracting to a good resoultion. All of them are salt based condition 
and I am not able to formulate a good cryoprotectant for these 
crystals. I also think that in my case the poor resolution is due to a 
poor cryoprotectant selection.


The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good 
diffraction with using lower peg and glycerol based cryoprotectants.


Can anybody suggest me good cryos conditions for salt based 
crystallization conditions or anything good for SaltRx crystallization 
hits.


Thanks

Firdous



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



--
Magnus Claesson, PhD
Lab manager, Drew-lab
Department of Biochemistry and Biophysics
Stockholm University




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Re: [ccp4bb] [EXTERNAL] [ccp4bb] suggestions for cryoprotectant

[Mooers, Blaine H.M. (HSC)]

Hi Firdous,

What Diana suggested may work if done carefully; however, I failed to transfer 
crystals from 1.5 LiSO4 salts to higher concentrations of LiSO4 in many 
attempts.
I did have success in transferring said crystals to 1.5 M sodium malonate then 
to incrementally to 2.8 or 3.2 M sodium malonate.

Best regards,

Blaine

Blaine Mooers, Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
College of Medicine
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center (BRC) Rm. 466
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419

office: (405) 271-8300   lab: (405) 271-8313
e-mail:  blaine-moo...@ouhsc.edu <mailto:blaine-moo...@ouhsc.edu>
alternate e-mail: bmooe...@gmail.com <mailto:bmooe...@gmail.com>

Faculty webpage: https://tinyurl.com/BMBmooers
Google Scholar: https://tinyurl.com/GSbmooers
SSRL User Executive Committee: 
https://www-ssrl.slac.stanford.edu/content/about-ssrl/advisory-panels/ssrl-users-organization
SSRL-LCLS User Meeting: https://conf.slac.stanford.edu/ssrl-lcls-2018/
EasyPyMOL: https://github.com/MooersLab
Molecular Graphics Course Links: https://tinyurl.com/molgr
Small Angle Scattering Links: https://tinyurl.com/ouhscsaxs
X-ray lab (LBSF): https://tinyurl.com/ouhsclbsf

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Firdous Tarique 
[kahkashantari...@gmail.com]
Sent: Friday, October 19, 2018 4:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] suggestions for cryoprotectant

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] suggestions for cryoprotectant

[Diana Tomchick]

Condition #3 isn’t far from the saturated conditions for lithium sulfate. 
Perhaps slightly increasing the lithium sulfate concentration will work, or 
using a related lithium salt.

See

Cryosalts: suppression of ice formation in macromolecular crystallography
Acta Cryst D
Volume 56| Part 
8| August 
2000| Pages 996-1001
doi:10.1107/S090744497587

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Oct 19, 2018, at 4:57 PM, Firdous Tarique 
mailto:kahkashantari...@gmail.com>> wrote:

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




UT Southwestern


Medical Center



The future of medicine, today.




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Re: [ccp4bb] suggestions for cryoprotectant

[Oganesyan, Vaheh]

Firdous,

Many crystallographers (if not all) went through case like you describe. So far 
you’ve got great suggestions that are worth trying. I’ll add one more from my 
own experience. In all cases I tried it appeared that addition of 0.5 to 1% v/v 
of glycerol or ethylene glycol to drop (either having it added to protein 
solution or to precipitant mix) doesn’t change “crystallizability” of the 
protein. However, that minor presence of the cryo agent in the solution where 
crystals grew allows increasing the concentration of cryo agent to 20-25% 
during harvesting, which should be enough for cryopreservation.

Good luck.


Regards,

Vaheh Oganesyan, PhD
www.medimmune.com



From: CCP4 bulletin board  On Behalf Of Firdous Tarique
Sent: Friday, October 19, 2018 5:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for cryoprotectant

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] suggestions for cryoprotectant

[Hargreaves, David]

Hi Firdous,

I had success last week with a condition containing 1.5M MgCl2 which I’d tried 
cryo-ing with high salt before and got no diffraction.
Adding dimethylsulphoxide to a final concentration of 10% v/v in the well 
solution worked well for me. DMSO was bound in the structure which can be a 
pest. Maybe other low mwt organics e.g. butane 2,3 diol, at around 10% might 
also work.
Good luck!

David


Dr. David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure & Biophysics
Dr David Hargreaves.
Office R1-09/Lab FL54
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telephone: +441223223546
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Please consider the environment before printing this e-mail



From: CCP4 bulletin board  On Behalf Of Firdous Tarique
Sent: 19 October 2018 22:58
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for cryoprotectant

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://clicktime.symantec.com/a/1/TbJtumBIrBbqsKBPLB8RzbEf5A_QNLos2DtakHWXv9o=?d=wNv6P2QmMKzJRIeGSIntutsyNaCQtLMHktF40wAsjjTH2HI5rmHrASaAsvJd9cmveOm7oSDcQCWe_-NYh_gnKDEvQa9vr4Wu-ljsQ-CiMgGUJGUx0jsbOF2jWPFJQxPgTC5c3ckj_TxBwOSRS7oF3Ktf2S03YFjc4YlxA5IeFkw44maexOt1xM5NkXgtwx-2M2eXZX4vgGIBLf7VND_mZ1AlxnhijOZ_HzauItrCZoQfMRw5XZrjW1XAa2gqm8lQuHRXcoCqQsJRKt0U9zZ8RGtyTJ-JT5ssezzeJXpBhZg3TtXc8SzxwLdf3z2hFXlUk6kk0CHNzHq_1BHsZ6xvjf40Ny5wX-Nu6PdByrujRz0ZFVSQmMPO4X1kiPZTSd4h_u2v64LDIt5oQN5K0NtxXkmi0Skwcg%3D%3D=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1>



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Re: [ccp4bb] suggestions for cryoprotectant

[James Gordon]

This kit from molecular dimensions allows you to test a variety for cryo 
protectants
https://www.moleculardimensions.com/products/3732-CryoProtX/

Best

James
Get Outlook for Android<https://aka.ms/ghei36>



From: CCP4 bulletin board  on behalf of Joseph Brock 

Sent: Sunday, October 21, 2018 2:08:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] suggestions for cryoprotectant

Dear Friduous,

This is also a good paper to look at once you know your crystals diffract at 
room temperature:
https://www.ncbi.nlm.nih.gov/pubmed/10944336

Many of the salts in your condition will be cryo-protective if you increase 
their concentration to saturated levels.

Cheers,
Joseph.


Joseph S. Brock | PhD
Researcher
Drew Lab
Department of Biochemistry and Biophysics
Stockholm University
From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Sunday, 21 October 2018 1:00:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 19 Oct 2018 to 20 Oct 2018 (#2018-267)

There are 5 messages totaling 760 lines in this issue.

Topics of the day:

  1. suggestions for cryoprotectant (4)
  2. RapiData at SSRL 2019



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--

Date:Fri, 19 Oct 2018 17:23:49 -0700
From:Christine Gee 
Subject: Re: suggestions for cryoprotectant

Dear Fridous

Have a look at https://www.ncbi.nlm.nih.gov/pubmed/14646118




<https://www.ncbi.nlm.nih.gov/pubmed/14646118>
Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown 
macromolecular crystals. - PubMed - NCBI


www.ncbi.nlm.nih.gov

Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2356-8. Epub 2003 Nov 
27.


Malonate as a cryoprotectant

Or https://www.ncbi.nlm.nih.gov/pubmed/20606259




<https://www.ncbi.nlm.nih.gov/pubmed/20606259>
Cryoprotection properties of salts of organic acids: a case study for a 
tetragonal crystal of HEW lysozyme. - PubMed - NCBI


www.ncbi.nlm.nih.gov

Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. doi: 
10.1107/S0907444910015416. Epub 2010 Jun 19.



Regards
Christine

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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--

Date:Sat, 20 Oct 2018 02:21:41 +
From:"Smith, Clyde" 
Subject: RapiData at SSRL 2019

Hi everyone,

Applications are now open for the 2019 edition of RapiData at SSRL. RapiData is 
a practical course in macromolecular X-ray diffraction data collection, data 
processing and structure solution. The aim of the RapiData course is to educate 
and train young scientists in data collection and processing methods at 
synchrotron beamlines, using state-of-the-art software and instrumentation. The 
course will be held at the Stanford Synchrotron Radiation Lightsource (SSRL) 
located on the SLAC National Accelerator Laboratory campus in Menlo Park, CA, 
from May 5-10 2019. Course organizers for 2019 are Silvia Russi, Clyde Smith 
and Jeney Wierman.

The course will comprise hands-on experiments at the SSRL beamlines, software 
tutorials, and lectures on the following topics:
Specimen preparation, Data collection strategies, X-ray light sources, X-ray 
detectors, Data reduction, Structure solution by MAD, SAD and Molecular 
Replacement, Complementary methods (spectroscopy and small angle scattering)

Please visit http://smb.slac.stanford.edu/news/rapidata/rapidata-2019/ and use 
the links on the "registrati

Re: [ccp4bb] suggestions for cryoprotectant

[Joseph Brock]

Dear Friduous,


This is also a good paper to look at once you know your crystals diffract at 
room temperature:

https://www.ncbi.nlm.nih.gov/pubmed/10944336


Many of the salts in your condition will be cryo-protective if you increase 
their concentration to saturated levels.


Cheers,

Joseph.


Joseph S. Brock | PhD
Researcher
Drew Lab
Department of Biochemistry and Biophysics
Stockholm University

From: CCP4 bulletin board  on behalf of CCP4BB automatic 
digest system 
Sent: Sunday, 21 October 2018 1:00:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: CCP4BB Digest - 19 Oct 2018 to 20 Oct 2018 (#2018-267)

There are 5 messages totaling 760 lines in this issue.

Topics of the day:

  1. suggestions for cryoprotectant (4)
  2. RapiData at SSRL 2019



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https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

--

Date:Fri, 19 Oct 2018 17:23:49 -0700
From:Christine Gee 
Subject: Re: suggestions for cryoprotectant

Dear Fridous

Have a look at https://www.ncbi.nlm.nih.gov/pubmed/14646118
[http://www.ncbi.nlm.nih.gov/coreutils/img/pubmed256blue.png]

Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown 
macromolecular crystals. - PubMed - 
NCBI
www.ncbi.nlm.nih.gov
Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2356-8. Epub 2003 Nov 
27.


Malonate as a cryoprotectant

Or https://www.ncbi.nlm.nih.gov/pubmed/20606259
[http://www.ncbi.nlm.nih.gov/coreutils/img/pubmed256blue.png]

Cryoprotection properties of salts of organic acids: a case study for a 
tetragonal crystal of HEW lysozyme. - PubMed - 
NCBI
www.ncbi.nlm.nih.gov
Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. doi: 
10.1107/S0907444910015416. Epub 2010 Jun 19.



Regards
Christine

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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--

Date:Sat, 20 Oct 2018 02:21:41 +
From:"Smith, Clyde" 
Subject: RapiData at SSRL 2019

Hi everyone,

Applications are now open for the 2019 edition of RapiData at SSRL. RapiData is 
a practical course in macromolecular X-ray diffraction data collection, data 
processing and structure solution. The aim of the RapiData course is to educate 
and train young scientists in data collection and processing methods at 
synchrotron beamlines, using state-of-the-art software and instrumentation. The 
course will be held at the Stanford Synchrotron Radiation Lightsource (SSRL) 
located on the SLAC National Accelerator Laboratory campus in Menlo Park, CA, 
from May 5-10 2019. Course organizers for 2019 are Silvia Russi, Clyde Smith 
and Jeney Wierman.

The course will comprise hands-on experiments at the SSRL beamlines, software 
tutorials, and lectures on the following topics:
Specimen preparation, Data collection strategies, X-ray light sources, X-ray 
detectors, Data reduction, Structure solution by MAD, SAD and Molecular 
Replacement, Complementary methods (spectroscopy and small angle scattering)

Please visit http://smb.slac.stanford.edu/news/rapidata/rapidata-2019/ and use 
the links on the "registration" page to apply for the course. The application 
deadline is December 31 2018 Successful applicants will be notified early in 
January 2019 and invited to register and book accommodation at the SLAC 
Guesthouse at that time. A limited amount of travel support funding may be 
available.

Re: [ccp4bb] suggestions for cryoprotectant

[Thomas, Leonard M.]

This is the first thing you need to do before going down the rabbit hole 
looking for a cryo protectant.  Mitegen make it much easier then in the 'old 
days' of capillaries.

Good luck,
Len

lmtho...@ou.edu

On Oct 20, 2018 2:02 AM, Andreas Förster  wrote:
Dear Firdous,

get some Mitegen sleeves and test your crystals at RT. Before you establish  
diffraction, there’s no need to agonize about cryo-conditions.

Good luck!


Andreas

On Fri, 19 Oct 2018 at 23:57, Firdous Tarique 
mailto:kahkashantari...@gmail.com>> wrote:
Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



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Re: [ccp4bb] suggestions for cryoprotectant

[mesters]

Hi,

have a look at the following interesting papers

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466044/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852300/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4252874/

Good luck

J.

Am 19.10.18 um 23:57 schrieb Firdous Tarique:

Dear members

I have got beautiful crystal hits in SaltRx screens which are not 
diffracting to a good resoultion. All of them are salt based condition 
and I am not able to formulate a good cryoprotectant for these 
crystals. I also think that in my case the poor resolution is due to a 
poor cryoprotectant selection.


The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good 
diffraction with using lower peg and glycerol based cryoprotectants.


Can anybody suggest me good cryos conditions for salt based 
crystallization conditions or anything good for SaltRx crystallization 
hits.


Thanks

Firdous



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Re: [ccp4bb] suggestions for cryoprotectant

[Andreas Förster]

Dear Firdous,

get some Mitegen sleeves and test your crystals at RT. Before you establish
 diffraction, there’s no need to agonize about cryo-conditions.

Good luck!


Andreas

On Fri, 19 Oct 2018 at 23:57, Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] suggestions for cryoprotectant

[Christine Gee]

Dear Fridous

Have a look at https://www.ncbi.nlm.nih.gov/pubmed/14646118
Malonate as a cryoprotectant

Or https://www.ncbi.nlm.nih.gov/pubmed/20606259

Regards
Christine

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] suggestions for cryoprotectant

[Pascal Egea]

Hi Firdous,

I noticed that conditions 2,4 and 5 are overall harsh agents (chaotropic
agents such as KSCN (probably not KCN) and Nitrate ions ), in my own
personal experience I have never been very lucky with crystals grown in
nitrate ions.
Condition 1 however looks a bit more soft with 'gentle' ions.
May I suggest trying a sodium malonate screen or seeding in such a
crystallization agent and maybe trying to cryprotect those acetate crystals
in malonate which is a cryo-friendly so-called 'magic salt'. crystals grown
in carboxylates such as citrate and malonate are usually easier to
cryo-protect.

Hope this helps

Pascal Egea

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


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[ccp4bb] suggestions for cryoprotectant

[Firdous Tarique]

Dear members

I have got beautiful crystal hits in SaltRx screens which are not
diffracting to a good resoultion. All of them are salt based condition and
I am not able to formulate a good cryoprotectant for these crystals. I also
think that in my case the poor resolution is due to a poor cryoprotectant
selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction
with using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



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Re: [ccp4bb] suggestions for cryoprotectant

[Enrico Stura]

Dear All,

Since 80% saturated Li2SO4 has not been mentioned, I will do so. It is a  
good cryosalt and I have often used

it even without any buffer added.
see:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth  Design, e-print

http://pubs.acs.org/doi/full/10.1021/cg301531f

The problem with phosphate precipitants is the formation of salt crystals  
in the cryoprotectant solution.
Cryoprotecting molecules that normally work well with high ionic strength  
conditions do not always

work well with phosphates.

800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic
is well matched by 80% saturated lithum sulphate in terms of ionic  
strength.


Enrico.


On Fri, 07 Feb 2014 06:20:58 +0100, Tanner, John J.  
tanne...@missouri.edu wrote:



Try L-proline.  It works well with high ionic strength conditions:

http://www.ncbi.nlm.nih.gov/pubmed/22868767

Sent from Jack's iPad

On Feb 6, 2014, at 10:40 PM, Deepak Thankappan Nair  
deepaktn...@gmail.commailto:deepaktn...@gmail.com wrote:


Hello,
Does anybody know what would be a good cryoprotectant for the following  
condition:
800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic  
100 mM Sodium acetate/Aceticacid pH4.5


Thanks
Deepak




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Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
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e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

[ccp4bb] suggestions for cryoprotectant

[Deepak Thankappan Nair]

Hello,
Does anybody know what would be a good cryoprotectant for the following
condition:
800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic 100
mM Sodium acetate/Aceticacid pH4.5

Thanks
Deepak

Re: [ccp4bb] suggestions for cryoprotectant

[Roger Rowlett]

Lots of choices. I usually try the crystallization solution + 30% glucose
first. Glycerol or ethylene glycol are other possibilities here. Another
possibility is 2.5-3.0 M sodium malonate at a similar pH.

Roger Rowlett
On Feb 6, 2014 11:40 PM, Deepak Thankappan Nair deepaktn...@gmail.com
wrote:

 Hello,
 Does anybody know what would be a good cryoprotectant for the following
 condition:
 800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic 100
 mM Sodium acetate/Aceticacid pH4.5

 Thanks
 Deepak

Re: [ccp4bb] suggestions for cryoprotectant

[Edward Snell]

You might get some clues from 

Acta Cryst. (2008). D64, 287-301[ doi:10.1107/S0907444907067613 ]
Glycerol concentrations required for the successful vitrification of cocktail 
conditions in a high-throughput crystallization screen
R. Kempkes, et al.

and 

J. Appl. Cryst. (2002). 35, 538-545[ doi:10.1107/S0021889802009238 ]
The development and application of a method to quantify the quality of 
cryoprotectant solutions using standard area-detector X-ray images
M. B. McFerrin and E. H. Snell

both shamelessly building on the original work

J. Appl. Cryst. (1996). 29, 584-587[ doi:10.1107/S0021889896004190 ]
Glycerol concentrations required for cryoprotection of 50 typical protein 
crystallization solutions
E. F. Garman and E. P. Mitchell

Most importantly check out

J. Appl. Cryst. (1994). 27, 1070-1074[ doi:10.1107/S0021889894008629 ]
Flash freezing of protein crystals: investigation of mosaic spread and 
diffraction limit with variation of cryoprotectant concentration
E. P. Mitchell and E. F. Garman

as the cryocondition that causes a successful vitrification may need to be 
optimized further to get the best diffraction.

Also - try room temp first so you know what your cryoprotectant is doing to 
your crystal.

PS. Given the discussions room temperature crystals and the synchrotron, if you 
do this, make sure you keep the stream blowing at room temp. It still helps to 
remove heat that builds up.

Cheers,

Eddie

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Deepak 
Thankappan Nair [deepaktn...@gmail.com]
Sent: Thursday, February 06, 2014 11:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for cryoprotectant

Hello,
Does anybody know what would be a good cryoprotectant for the following 
condition:
800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic 100 mM 
Sodium acetate/Aceticacid pH4.5

Thanks
Deepak

Re: [ccp4bb] suggestions for cryoprotectant

[Tanner, John J.]

Try L-proline.  It works well with high ionic strength conditions:

http://www.ncbi.nlm.nih.gov/pubmed/22868767

Sent from Jack's iPad

On Feb 6, 2014, at 10:40 PM, Deepak Thankappan Nair 
deepaktn...@gmail.commailto:deepaktn...@gmail.com wrote:

Hello,
Does anybody know what would be a good cryoprotectant for the following 
condition:
800 mM Sodium phosphate monobasic/1200 mM Potassium phosphate dibasic 100 mM 
Sodium acetate/Aceticacid pH4.5

Thanks
Deepak