Re: [ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Hi all

Just wanted to thank you all for all the suggestions and tell you of all
the things i tried what finally worked, if anyones interested.

Among the several things mentioned here, the crystals diffracted the best
(~ 2.9 Å at the synchrotron) when grown in a solution containing either 5%
ethylene glycol and cryo protected in 20% ethylene glycol or 15% glycerol
and protected in 25% glycerol.

Thanks for all the help

Mahesh


On Thu, Jan 23, 2014 at 7:04 AM, Elizabeth Morris <
emorr...@staffmail.ed.ac.uk> wrote:

> Hi Mahesh,
>
> I have experienced similar situations with AmSO4 as a precipitant. One
> thing I found worked is to optimise crystallization in a lower
> concentration of AmSO4 so that you reduce the amount of salt crystals you
> get when you try and loop your crystals. I got hits in 2 M AmSO4, but I
> have managed to reduce this to ~1 M and still get crystals by:
>
> - increasing protein concentration
> - varying pH/buffer
> - using microseeding to obtain crystals
>
> I have less experience with different types of cryoprotectants, but in my
> opinion, if you problem is that you see salt crystals when you open your
> droplet to the air, try reducing the salt concentration in your drop.
>
> Alternatively, perhaps your microscope is heating up your sample and
> increasing the rate of evaporation from your drop. If that is the case, try
> to work at lower intensities of the lamp, or turn it off more frequently.
>
> Finally, I have heard of people putting a damp tissue or a humidifier near
> to where you are looping your crystals to reduce evaporation of water from
> your drop.
>
> Good luck!
>
> Liz
>
>
>
>
>
>
> Quoting Mahesh Lingaraju  on Wed, 22 Jan 2014 12:28:15
> -0500:
>
>  Hello folks,
>>
>> I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
>> dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
>> finding the right cryoprotection for salt based conditions; I have tried
>> glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
>> ammonium sulphate concentration in presence of 5-10% glycerol. The
>> crystals
>> disintegrate in any kind of PEG based cryo. I made all these solutions in
>> the mother liquor and tested if they freeze clearly before using them.
>> However when I loop the crystals and try to soak them in these cryo-
>> mother
>> liquor, a lot of salt crystals suddenly form around the protein crystal
>> and
>> I see diffraction only from these salt crystals. The best I have been able
>> to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
>> cryoprotectant.
>>
>> I have also tried using 1 M sodium malonate as the cryoprotectant but my
>> crystals are not too stable in this mother liquor probably because I had
>> to
>> lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
>> crystals instantly when exposed to air.
>>
>> Other than trying to make the crystals more cryo-ready by finding other
>> hits or may be growing the same crystals with some cryoprotectant, I was
>> wondering if any of you have ideas based on your experience or suggestions
>> in case I am doing everything wrong in the first place.
>>
>> Thanks for all the help,
>>
>> Mahesh
>>
>>
>
>
> --
> The University of Edinburgh is a charitable body, registered in
> Scotland, with registration number SC005336.
>
>
>

[ccp4bb] cryoprotection condition

[rana ibd]

Dear CCP4
Thank you for your suggestions
Best Regards
Rana

Re: [ccp4bb] cryoprotection condition

[Matthias Zebisch]

Hi Rana,

you are almost there. For the first one I'd add (if at all) 5% PEG200 
and for the second I'd mix in 10% PEG200.

Only very big crystals might need even more cryoprotectant.

Best, Matthias


-
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK

Phone (+44) 1865 287549;
Fax (+44) 1865 287547
Email matth...@strubi.ox.ac.uk
Website http://www.strubi.ox.ac.uk
-

On 2/8/2014 12:44 PM, rana ibd wrote:

Dear CCP4
Does anyone know a good cryoprotection condition for these two 
conditions I would be grateful

The first condition:
 10% PEG 2, 20%PEG MME 550. 0.03M of NPS ( NPS is sodium nitrate, 
disodium hydrogen phosphate, and ammonium sulfate), 0.1MMES/Imadizole 
pH 6.5

The second condition:
20% PEG 3350, 0.2M sodium iodide or 0.2M sodium acetate
Thank you
Rana

[ccp4bb] cryoprotection condition

[rana ibd]

Dear CCP4
Does anyone know a good cryoprotection condition for these two conditions I 
would be grateful
The first condition:
 10% PEG 2, 20%PEG MME 550. 0.03M of NPS ( NPS is sodium nitrate, disodium 
hydrogen phosphate, and ammonium sulfate), 0.1MMES/Imadizole pH 6.5
The second condition:
20% PEG 3350, 0.2M sodium iodide or 0.2M sodium acetate
Thank you
Rana 

Re: [ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Thanks for all the suggestions. Ill try these and post an update here
regarding what happens.

Thank you so much !!

Mahesh


On Wed, Jan 22, 2014 at 1:23 PM, Mark van der Woerd
wrote:

> Mahesh,
>
> Have you tried to see what happens when you do not freeze the crystals at
> all? Do you get good resolution data? Do the crystals suffer radiation
> damage?
>
> Those are two important questions. First, if your crystals do not diffract
> well before you freeze them, usually (but not always) they will also not
> diffract well after you freeze them and you can try cryo-conditions until
> you see blue in the face, but they will never improve. In that case, you
> need new/better crystals. Second, many well-diffracting crystals do not
> necessarily need to be frozen at the home source. Of course they do need to
> be frozen at the synchrotron, but if you can get good (enough) data at
> home...
>
> On to recipes: try increasing dioxane. Try adding alcohols (somewhat
> similar to dioxane, maybe iso-propanol). Expect this to be a pain: very
> volatile agents make it difficult to harvest crystals. Of course try the
> mixtures without protein or crystals for freezing conditions and how well
> you can mix the components. Salt and organics are often not compatible.
>
> Try to regrow your crystals in sodium malonate (replace Ammonium sulfate
> with malonate). Try to regrow them in the presence of small amounts of the
> cryo-protectants you have already tried. For the ones that work, repeat
> with higher amounts. Very often crystals are much happier when you don't
> soak/tinker with them and freeze them straight out of the drop.
>
> Good luck.
>
> Mark
>
>
>
>  -Original Message-
> From: Mahesh Lingaraju 
> To: CCP4BB 
> Sent: Wed, Jan 22, 2014 10:38 am
> Subject: [ccp4bb] cryoprotection ideas for salt based condition
>
>  Hello folks,
>
>  I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
> dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
> finding the right cryoprotection for salt based conditions; I have tried
> glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
> ammonium sulphate concentration in presence of 5-10% glycerol. The crystals
> disintegrate in any kind of PEG based cryo. I made all these solutions in
> the mother liquor and tested if they freeze clearly before using them.
> However when I loop the crystals and try to soak them in these cryo- mother
> liquor, a lot of salt crystals suddenly form around the protein crystal and
> I see diffraction only from these salt crystals. The best I have been able
> to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
> cryoprotectant.
>
>  I have also tried using 1 M sodium malonate as the cryoprotectant but my
> crystals are not too stable in this mother liquor probably because I had to
> lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
> crystals instantly when exposed to air.
>
>  Other than trying to make the crystals more cryo-ready by finding other
> hits or may be growing the same crystals with some cryoprotectant, I was
> wondering if any of you have ideas based on your experience or suggestions
> in case I am doing everything wrong in the first place.
>
>  Thanks for all the help,
>
>  Mahesh
>

Re: [ccp4bb] cryoprotection ideas for salt based condition

[Mark van der Woerd]

Mahesh,

Have you tried to see what happens when you do not freeze the crystals at all? 
Do you get good resolution data? Do the crystals suffer radiation damage?

Those are two important questions. First, if your crystals do not diffract well 
before you freeze them, usually (but not always) they will also not diffract 
well after you freeze them and you can try cryo-conditions until you see blue 
in the face, but they will never improve. In that case, you need new/better 
crystals. Second, many well-diffracting crystals do not necessarily need to be 
frozen at the home source. Of course they do need to be frozen at the 
synchrotron, but if you can get good (enough) data at home...

On to recipes: try increasing dioxane. Try adding alcohols (somewhat similar to 
dioxane, maybe iso-propanol). Expect this to be a pain: very volatile agents 
make it difficult to harvest crystals. Of course try the mixtures without 
protein or crystals for freezing conditions and how well you can mix the 
components. Salt and organics are often not compatible. 

Try to regrow your crystals in sodium malonate (replace Ammonium sulfate with 
malonate). Try to regrow them in the presence of small amounts of the 
cryo-protectants you have already tried. For the ones that work, repeat with 
higher amounts. Very often crystals are much happier when you don't soak/tinker 
with them and freeze them straight out of the drop. 

Good luck.

Mark

 

 

 

-Original Message-
From: Mahesh Lingaraju 
To: CCP4BB 
Sent: Wed, Jan 22, 2014 10:38 am
Subject: [ccp4bb] cryoprotection ideas for salt based condition


Hello folks, 


I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10% dioxane 
and 1.6-2 M Ammonium sulphate. Based on little experience I have finding the 
right cryoprotection for salt based conditions; I have tried glycerol (15-25%), 
Ethylene glycol (20-25%), DMSO (15-20%) and increasing ammonium sulphate 
concentration in presence of 5-10% glycerol. The crystals disintegrate in any 
kind of PEG based cryo. I made all these solutions in the mother liquor and 
tested if they freeze clearly before using them. However when I loop the 
crystals and try to soak them in these cryo- mother liquor, a lot of salt 
crystals suddenly form around the protein crystal and I see diffraction only 
from these salt crystals. The best I have been able to get so far is ~ 9Å 
diffraction (Home-source) with DMSO as the cryoprotectant.


I have also tried using 1 M sodium malonate as the cryoprotectant but my 
crystals are not too stable in this mother liquor probably because I had to 
lower the ammonium sulphate by ~ 7-10% to make the drop not form salt crystals 
instantly when exposed to air. 


Other than trying to make the crystals more cryo-ready by finding other hits or 
may be growing the same crystals with some cryoprotectant, I was wondering if 
any of you have ideas based on your experience or suggestions in case I am 
doing everything wrong in the first place.


Thanks for all the help, 


Mahesh  

Re: [ccp4bb] cryoprotection ideas for salt based condition

[Roger Rowlett]

Mahesh,

Try 25-30% glucose. You can gradually add well solution + 37.5-40% 
glucose directly to the drop if your crystals are sensitive to changes 
in osmotic pressure. If your drop is evaporating too quickly, try 
working in the cold room or under oil to slow down evaporation. You can 
find our standard cryoprotection protocol on our wiki here 
 
under "No-fail" cryoprotection.


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 1/22/2014 12:28 PM, Mahesh Lingaraju wrote:

Hello folks,

I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10% 
dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I 
have finding the right cryoprotection for salt based conditions; I 
have tried glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) 
and increasing ammonium sulphate concentration in presence of 5-10% 
glycerol. The crystals disintegrate in any kind of PEG based cryo. I 
made all these solutions in the mother liquor and tested if they 
freeze clearly before using them. However when I loop the crystals and 
try to soak them in these cryo- mother liquor, a lot of salt crystals 
suddenly form around the protein crystal and I see diffraction only 
from these salt crystals. The best I have been able to get so far is ~ 
9Å diffraction (Home-source) with DMSO as the cryoprotectant.


I have also tried using 1 M sodium malonate as the cryoprotectant but 
my crystals are not too stable in this mother liquor probably because 
I had to lower the ammonium sulphate by ~ 7-10% to make the drop not 
form salt crystals instantly when exposed to air.


Other than trying to make the crystals more cryo-ready by finding 
other hits or may be growing the same crystals with some 
cryoprotectant, I was wondering if any of you have ideas based on your 
experience or suggestions in case I am doing everything wrong in the 
first place.


Thanks for all the help,

Mahesh

[ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Hello folks,

I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
finding the right cryoprotection for salt based conditions; I have tried
glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
ammonium sulphate concentration in presence of 5-10% glycerol. The crystals
disintegrate in any kind of PEG based cryo. I made all these solutions in
the mother liquor and tested if they freeze clearly before using them.
However when I loop the crystals and try to soak them in these cryo- mother
liquor, a lot of salt crystals suddenly form around the protein crystal and
I see diffraction only from these salt crystals. The best I have been able
to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
cryoprotectant.

I have also tried using 1 M sodium malonate as the cryoprotectant but my
crystals are not too stable in this mother liquor probably because I had to
lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
crystals instantly when exposed to air.

Other than trying to make the crystals more cryo-ready by finding other
hits or may be growing the same crystals with some cryoprotectant, I was
wondering if any of you have ideas based on your experience or suggestions
in case I am doing everything wrong in the first place.

Thanks for all the help,

Mahesh

[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] cryoprotection

[Herman . Schreuder]

Dear Enrico,

You are right that the trick has its limitations and I am aware of it. However, 
it might not be as bad as you think. Fiddling with the crystallization buffer 
or transferring crystals to different buffers also causes stress to the 
crystals and in many cases loss of resolution. If it turns out that the 
reservoir solution freezes ok (or the crystallization drop itself if that is 
feasible), I would risk, as I said, trying to freeze a crystal directly from 
the drop without any further manipulations. Why try to cryoprotect a crystal 
when it is not necessary? If that does not work, I would go for more elaborate 
protocols, which, as far as I know, also do not have 100% guarantee of success. 
In that case I would also consult the literature like the excellent paper you 
mention. 

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Enrico 
Stura
Gesendet: Dienstag, 27. August 2013 11:58
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] AW: [ccp4bb] cryoprotection

Herman,

The trick you suggest is not as valid as you may think. The ice rings can 
originate from the crystal itself.
If you crystallize in a high concentration PEG precipitant you will avoid ice 
rings, but if you transfer or soak your crystals in the same solution the high 
molecular weight PEG will not enter the crystal lattice and you will still get 
ice rings.
I have a picture of this in:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth & Design, e-print
http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G.

So cryoprotectants need to penetrate the crystal lattice to prevent ice rings, 
but even in the presence of ice rings the data can be used.

Regarding optimization:
The main problem you encounter in cryoprotection is that some compounds like 
glycerol and ethylene glycol solubilize protein crystals, but if you create a 
mixture of various compounds that is precipitation-solubilization neutral, then 
there is no real need for optimization.

Enrico.

On Tue, 27 Aug 2013 08:30:28 +0200,  wrote:

> A trick I like is just to freeze the reservoir solution or would-be 
> cryo-solution without a crystal present. If the frozen solution stays 
> clear and does not show ice rings on e.g.  a home source, it is worth 
> trying. Otherwise, the solution needs optimization.
> Herman
>
>
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
> Uday Kumar
> Gesendet: Freitag, 23. August 2013 19:52
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [ccp4bb] cryoprotection
>
> Hello
>
> Can anyone suggest a cryoprotectant for the following crystallization 
> condition
>
> 0.2-0.4M sodium formate
>
> ~20% PEG 3350
>
> 0-25 mM Nickel
>
> 0-100 mM Malonate
>
> Thank you
>
> with regards
> uday


-- 
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=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

Re: [ccp4bb] AW: [ccp4bb] cryoprotection

[Enrico Stura]

Herman,

The trick you suggest is not as valid as you may think. The ice rings can  
originate from the crystal itself.
If you crystallize in a high concentration PEG precipitant you will avoid  
ice rings,
but if you transfer or soak your crystals in the same solution the high  
molecular weight

PEG will not enter the crystal lattice and you will still get ice rings.
I have a picture of this in:
Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography.  
Crystal Growth & Design, e-print

http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G.

So cryoprotectants need to penetrate the crystal lattice to prevent ice  
rings, but even in the

presence of ice rings the data can be used.

Regarding optimization:
The main problem you encounter in cryoprotection is that some compounds
like glycerol and ethylene glycol solubilize protein crystals, but if you  
create a
mixture of various compounds that is precipitation-solubilization neutral,  
then

there is no real need for optimization.

Enrico.

On Tue, 27 Aug 2013 08:30:28 +0200,  wrote:

A trick I like is just to freeze the reservoir solution or would-be  
cryo-solution without a crystal present. If the frozen solution stays  
clear and does not show ice rings on e.g.  a home source, it is worth  
trying. Otherwise, the solution needs optimization.

Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von  
Uday Kumar

Gesendet: Freitag, 23. August 2013 19:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] cryoprotection

Hello

Can anyone suggest a cryoprotectant for the following crystallization  
condition


0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday



--
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=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

[ccp4bb] AW: [ccp4bb] cryoprotection

[Herman . Schreuder]

A trick I like is just to freeze the reservoir solution or would-be 
cryo-solution without a crystal present. If the frozen solution stays clear and 
does not show ice rings on e.g.  a home source, it is worth trying. Otherwise, 
the solution needs optimization.
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Uday 
Kumar
Gesendet: Freitag, 23. August 2013 19:52
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] cryoprotection

Hello

Can anyone suggest a cryoprotectant for the following crystallization condition

0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday

Re: [ccp4bb] cryoprotection

[Reddiravikumar Kumar]

Dear uday,

I have grown crystals in 0.2M sodium formate and 20% peg3350, i tried cryo
solution cotaining 0.2M sodium formate and peg1050 40%, it worked well for
me and diffracted to 2.3A at home source.
0.2-0.4M sodium formate

40% PEG 1050

0-25 mM Nickel

0-100 mM Malonate


On Fri, Aug 23, 2013 at 11:46 PM, Alexander D. Scouras  wrote:

> I've had good success transferring crystals grown in
>
> 200 mM NaOAc pH 5.5 / 10% PEG
>
> Into a solution for room temperature diffraction (in ~6 steps, peg first,
> then buffers) :
>
> 100 mM NaOAc / 100 mM Malonate / 30% PEG
>
> And from there into a cryo solution in 4-5 steps) that was:
>
> 100 mM NaOAc / 100 mM Malonate / 30% PEG / 20% Ethylene Glycol
>
>
> Crystals have been solved to at least 1.4A resolution with mosaicity in
> the 0.2-0.3 range.
>
> -Alex
>
>
>
>
> On Aug 23, 2013, at 10:52 AM, Uday Kumar  wrote:
>
> > Hello
> >
> > Can anyone suggest a cryoprotectant for the following crystallization
> condition
> >
> > 0.2-0.4M sodium formate
> >
> > ~20% PEG 3350
> >
> > 0-25 mM Nickel
> >
> > 0-100 mM Malonate
> >
> > Thank you
> >
> > with regards
> > uday
>



-- 
ravi kumar

Re: [ccp4bb] cryoprotection

[Alexander D. Scouras]

I've had good success transferring crystals grown in

200 mM NaOAc pH 5.5 / 10% PEG 

Into a solution for room temperature diffraction (in ~6 steps, peg first, then 
buffers) :

100 mM NaOAc / 100 mM Malonate / 30% PEG

And from there into a cryo solution in 4-5 steps) that was:

100 mM NaOAc / 100 mM Malonate / 30% PEG / 20% Ethylene Glycol


Crystals have been solved to at least 1.4A resolution with mosaicity in the 
0.2-0.3 range. 

-Alex




On Aug 23, 2013, at 10:52 AM, Uday Kumar  wrote:

> Hello
> 
> Can anyone suggest a cryoprotectant for the following crystallization 
> condition
> 
> 0.2-0.4M sodium formate
> 
> ~20% PEG 3350
> 
> 0-25 mM Nickel
> 
> 0-100 mM Malonate
> 
> Thank you
> 
> with regards
> uday

Re: [ccp4bb] cryoprotection

[Bosch, Juergen]

increase your PEG3350 >27% and keep the other components at their current 
concentration. You can also add glycerol or ethylene glycol into the mix.

If you have multiple crystals then try various variants.

Jürgen

On Aug 23, 2013, at 1:52 PM, Uday Kumar wrote:

Hello

Can anyone suggest a cryoprotectant for the following crystallization condition

0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

[ccp4bb] cryoprotection

[Uday Kumar]

Hello

Can anyone suggest a cryoprotectant for the following crystallization condition

0.2-0.4M sodium formate

~20% PEG 3350

0-25 mM Nickel

0-100 mM Malonate

Thank you

with regards
uday

Re: [ccp4bb] cryoprotection for fragile crystals

[Sean Seaver]

I believe this is the article that you are looking for:

http://www.xtals.org/crystal_cryo.pdf

Cheers,

Sean

[ccp4bb] cryoprotection for fragile crystals

[Laurie Betts]

I found within the last few years a nice flowchart that someone (I think it
was Artem Evdokimov) had posted somewhere for a protocol for dealing with
crystals that you couldn't just quick dip, and now I can't find it - maybe
he can send it to me offbb???  Thanks

Laurie Betts
UNC CHapel Hill

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Joerg Standfuss]

Hi Brenda,

Maybe you want to have a look at the cryoprotectant database for  
protein crystals.


http://idb.exst.jaxa.jp/db_data/protein/search-e.php

There you can search for your precipitant and get a list of  
compatible freezing conditions that worked before including the  
reference. It is a nice starting point and a good inspiration.


Joerg


On 19 Aug 2009, at 17:18, Schulman, Brenda wrote:


Hello!

I would be grateful for suggestions on cryoprotectants for crystals  
growing

in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer


***
Jörg Standfuss
Medical Research Council
Laboratory of Molecular Biology
Hills Road
Cambridge CB2 0QH

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Richard Bayliss]

I third it - we used 3.2M + 5% glycerol ammonium sulfate to  
cryoprotect crystals grown in 1.2M ammonium suphate (Bayliss et al.  
JBC 2002). This really helped diffraction too. My tip when handling  
drops at very high salt is to make a little circle of wet tissue paper  
around your coverslip to make a humidity chamber. You'll have much  
longer to fish crystals out of the drop before salt crystals start to  
form.


Good luck
Richard


Dr Richard Bayliss
Royal Society University Research Fellow
& Career Development Team Leader
Section of Structural Biology
Institute of Cancer Research
237 Fulham Road
London SW3 6JB
UK

Tel: +44 (0)20 71535557
Fax: +44 (0)20 71535457




On 19 Aug 2009, at 7:20 PM, James Holton wrote:

I second that one.  Ammonium sulfate is one of my favorite cryos.  I  
recommend making up a saturated solution of ammonium sulfate, as it  
is actually a very good cryo all by itself, and then "dilute" it by  
adding the rest of the stuff in your condition (buffers, etc. and a  
little bit of water).  You want to be a little below saturation so  
that the salt does not grow crystals of its own while you are  
soaking.  This is especially important if your protein crystals are  
hexagonal rods!


-James Holton
MAD Scientist

Savvas Savvides wrote:

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great  
succes by
cryo-cooling xtals grown at 3.2M AS straight out of their  
crystallization
drops. You can consult the "M&M" section in Kyndt J. et al  
Biochemistry 2007

Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas

 Savvas Savvides L-ProBE, Unit for Structural Biology Ghent  
University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32- 
(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be 
 http://www.lprobe.ugent.be/xray.html




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals  
growing

in > 3M ammonium sulfate.

Thanks!

Brenda


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Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[James Holton]

I second that one.  Ammonium sulfate is one of my favorite cryos.  I 
recommend making up a saturated solution of ammonium sulfate, as it is 
actually a very good cryo all by itself, and then "dilute" it by adding 
the rest of the stuff in your condition (buffers, etc. and a little bit 
of water).  You want to be a little below saturation so that the salt 
does not grow crystals of its own while you are soaking.  This is 
especially important if your protein crystals are hexagonal rods!


-James Holton
MAD Scientist

Savvas Savvides wrote:

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great succes by
cryo-cooling xtals grown at 3.2M AS straight out of their crystallization
drops. You can consult the "M&M" section in Kyndt J. et al Biochemistry 2007
Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer





E-mail message checked by Spyware Doctor (6.1.0.447)
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Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Brett, Thomas]

Or saturated LiSO4 works too. Should work in this case.
-Tom

Tom J. Brett, PhD
Assistant Professor of Medicine
Division of Pulmonary and Critical Care
Washington University School of Medicine
Campus Box 8052, 660 S. Euclid
Saint Louis, MO 63110

From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Savvas Savvides 
[savvas.savvi...@ugent.be]
Sent: Wednesday, August 19, 2009 11:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great succes by
cryo-cooling xtals grown at 3.2M AS straight out of their crystallization
drops. You can consult the "M&M" section in Kyndt J. et al Biochemistry 2007
Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas


Savvas Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19
Email: savvas.savvi...@ugent.be
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer





E-mail message checked by Spyware Doctor (6.1.0.447)
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Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Ohren, Jeffrey]

Dear Brenda,

I'd suggest using a mixture of 75% Paratone-N oil with 25% light, white
mineral oil. Mix the two thoroughly with a positive displacement pipette
then add a small amount next to the mother liquor (ML) drop. Remove a
crystal from the ML using a matched cryo loop (the smaller the better)
and then dip it completely into the oil. Gently slide the side of the
loop containing the crystal against the surface beside the ML to wick
off some of the oil, which will also remove some of the aqueous layer on
the crystal surface. Then dip the crystal back into the oil; it usually
takes 2-3 times to completely remove the aqueous layer. This works about
80% of the time for me for crystals grown from high salt conditions.
Method courtesy of A. McPherson and J. Pflugrath. 

HTH,
Jeff

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 12:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals
growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Savvas Savvides]

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great succes by
cryo-cooling xtals grown at 3.2M AS straight out of their crystallization
drops. You can consult the "M&M" section in Kyndt J. et al Biochemistry 2007
Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas

 
Savvas Savvides 
L-ProBE, Unit for Structural Biology 
Ghent University 
K.L. Ledeganckstraat 35 
9000 Ghent, BELGIUM 
office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 
Email: savvas.savvi...@ugent.be 
http://www.lprobe.ugent.be/xray.html



-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer





E-mail message checked by Spyware Doctor (6.1.0.447)
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Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Christine Gee]

Dear Brenda

Check out malonate
http://www.ncbi.nlm.nih.gov/pubmed/14646118? 
ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubm 
ed_DefaultReportPanel.Pubmed_RVDocSum


Malonate: a versatile cryoprotectant and stabilizing solution for  
salt-grown macromolecular crystals.

Holyoak T, Fenn TD, Wilson MA, Moulin AG, Ringe D, Petsko GA.

Regards
Christine


On 19/08/2009, at 9:18 AM, Schulman, Brenda wrote:


Hello!

I would be grateful for suggestions on cryoprotectants for crystals  
growing

in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Roger Rowlett]

Soluble possibilities include glucose, ethylene glycol, DMSO, glycerol 
(15-30%). You may not need much cryoprotectant at all at these salt 
concentrations. PEGs are not very soluble in high ammonium sulfate 
solutions. (I think PEG-400 can go up to only 4% or so at 2 M ammonium 
sulfate.)


Cheers.


Schulman, Brenda wrote:

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer
  

--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
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...@mail.colgate.edu

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Pascal Egea]

Hi Brenda,
You can try sugars like glucose, trehalose and sucrose for high AS contents.
It has been succesfully used in really hard cases such at protein RNA
crystals grown in AS.  see Acta Cryst (2002) D58 1664-1669 Garber et al.

HTH

Pascal Egea

[ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Schulman, Brenda]

Hello!

I would be grateful for suggestions on cryoprotectants for crystals growing
in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer