Re: [ccp4bb] Help needed finding hit condition

[khaja faisal tarique]

Hello everyone.

I remember the screen was again from Jenabioscience and this had happened
with one of my protein. The screen was very old and the condition was
peg3350, tris pH 8, lithium sulfate and NaCl as the salt. Hit was obtained
which was never reproducible. Luckily I solved the structure from the hit
itself which diffracted to 2.2A resolution. But it is still a mystery for
us but this is all crystallography is. Strange things happen.

Faisal
Postdoc
PHRI, NJ, USA

On Jul 31, 2017 5:19 PM, "Janet Newman"  wrote:

> ​Hi Jonathan,
>
>
> Hopefully you know about the trick of making any precious condition last
> longer - use the 'magic' solution only in the drop itself, and use the best
> approximation you can make in the reservoir of the experiment (if you are
> doing vapour diffusion)
>
>
> Regards, Janet
>
>
> Janet Newman
> Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
> CSIRO Material Science and Engineering
> 343 Royal Parade
> Parkville.  VIC. 3052
> Australia
> Tel +613 9662 7326 <+61%203%209662%207326>
> Email janet.new...@csiro.au
> --
> *From:* CCP4 bulletin board  on behalf of Jonathan
> Bailey 
> *Sent:* 31 July 2017 22:34
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Help needed finding hit condition
>
>
> Dear CCP4bb community
>
>
> I apologies for the slightly off topic post.
>
>
> We have recently had success crystallizing a membrane protein (diffraction
> > 3 Å at a synchrotron source) using the *in meso* method, the hit
> condition was from the Jena Bioscience screen Pi-minimal condition number
> #57.
>
>
> Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium
> bromide
>
>
> The screen is old and expired 12/20/2013 (lot # JBS00013133), we have
> tried to reproduce the crystals using homemade optimization screens around
> the hit condition but have not had any success. We have tried reproducing
> the hit using a new (not expired) Pi-minimal screen but had no success. We
> are only able to reproduce the crystals using the expired screen and we do
> not have much of it left.
>
>
>
> We went back and tested the pH of the condition that had given crystals,
> the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator
> strip. We believe the drop in pH is caused by oxidative degradation of the
> PEG1000 resulting in the formation of carboxylic acid species.
>
>
> We have contacted Jena Bioscience to try and get some of the old screen
> stock but unfortunately they do not have any.
>
>
> My question is does anyone out there happen to have any expired screen
> stocks of this Pi-minimal condition (#57), ideally from the same lot (lot #
> JB200013133), that they would be willing to send us.
>
>
>
> Does anyone have any advice as to how to reproduce the condition? We’ve
> considered bubbling oxygen through and heating the sample to accelerate the
> oxidation process.
>
>
>
> King Regards
>
>
> Jonathan Bailey (PhD student)
>
>
> Professor Martin Caffrey Lab MS group Trinity College Dublin
>

Re: [ccp4bb] Help needed finding hit condition

[Janet Newman]

?Hi Jonathan,


Hopefully you know about the trick of making any precious condition last longer 
- use the 'magic' solution only in the drop itself, and use the best 
approximation you can make in the reservoir of the experiment (if you are doing 
vapour diffusion)


Regards, Janet


Janet Newman
Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
CSIRO Material Science and Engineering
343 Royal Parade
Parkville.  VIC. 3052
Australia
Tel +613 9662 7326
Email janet.new...@csiro.au

From: CCP4 bulletin board  on behalf of Jonathan Bailey 

Sent: 31 July 2017 22:34
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Help needed finding hit condition

Dear CCP4bb community

I apologies for the slightly off topic post.

We have recently had success crystallizing a membrane protein (diffraction > 3 
Å at a synchrotron source) using the in meso method, the hit condition was from 
the Jena Bioscience screen Pi-minimal condition number #57.

Hit condition - 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium bromide

The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried to 
reproduce the crystals using homemade optimization screens around the hit 
condition but have not had any success. We have tried reproducing the hit using 
a new (not expired) Pi-minimal screen but had no success. We are only able to 
reproduce the crystals using the expired screen and we do not have much of it 
left.

We went back and tested the pH of the condition that had given crystals, the 
expected pH was 7.9 but we found it to be 6 - 6.5 using a pH indicator strip. 
We believe the drop in pH is caused by oxidative degradation of the PEG1000 
resulting in the formation of carboxylic acid species.

We have contacted Jena Bioscience to try and get some of the old screen stock 
but unfortunately they do not have any.

My question is does anyone out there happen to have any expired screen stocks 
of this Pi-minimal condition (#57), ideally from the same lot (lot # 
JB200013133), that they would be willing to send us.

Does anyone have any advice as to how to reproduce the condition? We've 
considered bubbling oxygen through and heating the sample to accelerate the 
oxidation process.

King Regards

Jonathan Bailey (PhD student)

Professor Martin Caffrey Lab MS group Trinity College Dublin

Re: [ccp4bb] Help needed finding hit condition

[Kevin Jin]

Hi Jonathan,

 Old buffer may have slow evaporation with some side reactions. The
concentration of each component may increase a little bit.  In this case,
 I would consider the condition as the origin for further optimization.
Each component may need to be considered separately.

For 150mM Tris pH 8.000,
The pH and concentration may  have a slight change (increase ?).  I will
try the concentration of 150mM, 155 mM and 160mM. For pH,  pH 8.1 and pH
8.2...

For KBr, the concentration may be 80, 85, 90mM or higher.

For 47.1% w/v PEG1K (Pretty high concentration),
As the result for ring-opening epoxide reaction, it is not very stable
anyway. The reaction always continued. Basic condition made the case even
worse. In this case, the Mn (MW) of PEG1K may not be that average anymore.
It is very possible that the polymer chain is elongated. Of course, the
concentration of PEG is increased too. In the meanwhile, the presence of
KBr may cause further chemical modification on the PEG chains.  You may try
PEG 95--1050 (Sigma P3515), PEG 1305-1595 (Sigma 202136), PEG3K or PEG
3350, etc.


Best,

Kevin

P.S. If you take a look from the top of your old solution in the tube,
what's the color? Slight Yellow? You can use a tube with dd water a
reference.


On Mon, Jul 31, 2017 at 5:34 AM, Jonathan Bailey  wrote:

> Dear CCP4bb community
>
>
> I apologies for the slightly off topic post.
>
>
> We have recently had success crystallizing a membrane protein (diffraction
> > 3 Å at a synchrotron source) using the *in meso* method, the hit
> condition was from the Jena Bioscience screen Pi-minimal condition number
> #57.
>
>
> Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium
> bromide
>
>
> The screen is old and expired 12/20/2013 (lot # JBS00013133), we have
> tried to reproduce the crystals using homemade optimization screens around
> the hit condition but have not had any success. We have tried reproducing
> the hit using a new (not expired) Pi-minimal screen but had no success. We
> are only able to reproduce the crystals using the expired screen and we do
> not have much of it left.
>
>
>
> We went back and tested the pH of the condition that had given crystals,
> the expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator
> strip. We believe the drop in pH is caused by oxidative degradation of the
> PEG1000 resulting in the formation of carboxylic acid species.
>
>
> We have contacted Jena Bioscience to try and get some of the old screen
> stock but unfortunately they do not have any.
>
>
> My question is does anyone out there happen to have any expired screen
> stocks of this Pi-minimal condition (#57), ideally from the same lot (lot #
> JB200013133), that they would be willing to send us.
>
>
>
> Does anyone have any advice as to how to reproduce the condition? We’ve
> considered bubbling oxygen through and heating the sample to accelerate the
> oxidation process.
>
>
>
> King Regards
>
>
> Jonathan Bailey (PhD student)
>
>
> Professor Martin Caffrey Lab MS group Trinity College Dublin
>



-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

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

Re: [ccp4bb] Help needed finding hit condition

[R. Michael Garavito]

Jonathan,

While your claim of oxidative degradation of PEG1000 may be true -- I gather 
you mean that the conversion of the ends of the PEG polymers to aldehydes or 
peroxides, then to carboxylates --  you should check out Fran Jurnak’s old 
paper (Journal of Crystal Growth 76, 577, 1986).  The synthesis of PEG often 
contains some of phosphoric acid due to the way they terminated the chain 
elongation.  It is variable from batch to batch and from supplier to supplier; 
Merck (Germany) is a fairly good source, but Baker/Union Carbide isn't.   
Unless Jena took the time and effort to purify the PEG (see Bill Ray's article 
in the same issue, p. 562), you have another factor that can drop the pH.  You 
might ask where Jena buys their PEG stock.

Also, the way many companies make the screens are not always clear.  Some just 
mix stocks, meaning the pH of the Tris stock (perhaps at 1 M) was pH 8, but 
when diluted down with the other components to make the 150 mM concentration 
for the screen mixture, the pH can be significantly different.  The dilution of 
Tris would drop the pH.

Cheers,

Michael
 

R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





> On Jul 31, 2017, at 8:34 AM, Jonathan Bailey  wrote:
> 
> Dear CCP4bb community
> 
> 
> 
> I apologies for the slightly off topic post.
> 
> 
> 
> We have recently had success crystallizing a membrane protein (diffraction > 
> 3 Å at a synchrotron source) using the in meso method, the hit condition was 
> from the Jena Bioscience screen Pi-minimal condition number #57.
> 
> 
> 
> Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium 
> bromide
> 
> 
> 
> 
> The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried 
> to reproduce the crystals using homemade optimization screens around the hit 
> condition but have not had any success. We have tried reproducing the hit 
> using a new (not expired) Pi-minimal screen but had no success. We are only 
> able to reproduce the crystals using the expired screen and we do not have 
> much of it left.
> 
>   
> We went back and tested the pH of the condition that had given crystals, the 
> expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator strip. 
> We believe the drop in pH is caused by oxidative degradation of the PEG1000 
> resulting in the formation of carboxylic acid species.
> 
> 
> 
> We have contacted Jena Bioscience to try and get some of the old screen stock 
> but unfortunately they do not have any.
> 
> 
> 
> My question is does anyone out there happen to have any expired screen stocks 
> of this Pi-minimal condition (#57), ideally from the same lot (lot # 
> JB200013133), that they would be willing to send us.
> 
>   
> Does anyone have any advice as to how to reproduce the condition? We’ve 
> considered bubbling oxygen through and heating the sample to accelerate the 
> oxidation process.
> 
>   
> King Regards
> 
> 
> 
> Jonathan Bailey (PhD student) 
> 
> 
> 
> Professor Martin Caffrey Lab MS group Trinity College Dublin 
> 

Re: [ccp4bb] Help needed finding hit condition

[Vellieux Frédéric]

Hello,

Such pH drifts are rather common with crystallisation screens. Have you tried 
to produce other precipitant solutions with ca. 47% PEG 1000, 80 mM Potassium 
bromide but having a pH of (say) 6.2 ? A pH rangle close to that where your 
crystals were obtained. This would mean changing buffering agent to… MES 
perhaps ?

Cheers,

Fred.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jonathan 
Bailey
Sent: Monday, July 31, 2017 2:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Help needed finding hit condition

Dear CCP4bb community

I apologies for the slightly off topic post.

We have recently had success crystallizing a membrane protein (diffraction > 3 
Å at a synchrotron source) using the in meso method, the hit condition was from 
the Jena Bioscience screen Pi-minimal condition number #57.

Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium bromide

The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried to 
reproduce the crystals using homemade optimization screens around the hit 
condition but have not had any success. We have tried reproducing the hit using 
a new (not expired) Pi-minimal screen but had no success. We are only able to 
reproduce the crystals using the expired screen and we do not have much of it 
left.

We went back and tested the pH of the condition that had given crystals, the 
expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator strip. 
We believe the drop in pH is caused by oxidative degradation of the PEG1000 
resulting in the formation of carboxylic acid species.

We have contacted Jena Bioscience to try and get some of the old screen stock 
but unfortunately they do not have any.

My question is does anyone out there happen to have any expired screen stocks 
of this Pi-minimal condition (#57), ideally from the same lot (lot # 
JB200013133), that they would be willing to send us.

Does anyone have any advice as to how to reproduce the condition? We’ve 
considered bubbling oxygen through and heating the sample to accelerate the 
oxidation process.

King Regards

Jonathan Bailey (PhD student)

Professor Martin Caffrey Lab MS group Trinity College Dublin
-

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