Re: [ccp4bb] [EXT] Re: [ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear Patrick,  

Thank you for the suggestions and for the detailed the statistics!! 

Regards 

Kavya 

On 2024-02-06 23:06, Patrick Shaw Stewart wrote:

> Hi Kavya 
> 
> 1. Make a seed stock from the globules or anything else that you think might 
> be crystalline, and recreen.  In other words, you should add your seed stock 
> to _random screens_ (not optimization experiments).  There could be many 
> conditions that are in the metastable zone of the phase diagram in your 
> normal screens - this method can give you crystals in those conditions. 
> 
> If this works, you'll be in a better position anyway because you'll have more 
> control - by diluting the seed stock, you can control the number of crystals 
> per drop. 
> 
> References: 
> 
>> D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed 
>> matrix-screening method for protein crystallization. _Acta Crystallographica 
>> Section D: Biological Crystallography_, _63_(4), pp.550-554. 
>> 
>> Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, 
>> P.F., 2011. Random microseeding: a theoretical and practical exploration of 
>> seed stability and seeding techniques for successful protein 
>> crystallization. _Crystal Growth & Design_, _11_(8), pp.3432-3441.
> 
> This is how we normally make the seed: 
> 
>> https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf
> 
> 2. Many years ago, we did some data mining of the crystallization conditions 
> in a remark in the PDB.  The concentrations that people reported are below.  
> There were eight reports where over 100 mg/mL was used.  It only goes up to 
> 2004. 
> 
>> https://www.douglas.co.uk/PDB_data.htm [1]
> 
> Good luck, 
> 
> Patrick 
> 
> On Mon, Feb 5, 2024 at 10:27 AM kavyashreem  
> wrote: 
> 
>> Dear All,  
>> 
>> Has anyone worked on a protein which is highly soluble even at 80mg/ml? 
>> 
>> We have one such candidate, which does not precipitate even at 80mg/ml 
>> instead forms phase separated globules in crystallization plate, which 
>> eventually hardens over a period of 1 to 1.5 months (which is florescent 
>> under UV microscope.) 
>> 
>> We tried screening at different pH, but failed to get any hits. 
>> 
>> Since we got few conditions in which the phase separated globules 
>> solidified, we focused on them and expanded with 120mg/ml protein, still 
>> there were not visible precipitates except for the phase separation. This 
>> has been a challenging target so far. We have tried with different 
>> constructs, which unfortunately are not soluble! 
>> 
>> Does POMs help in such cases? Or do you have any other suggestion.  
>> 
>> Thank you  
>> 
>> Regards 
>> 
>> Kavya 
>> 
>> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
>> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
>> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE 
>> SENDER IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT 
>> AUTHORISED TO COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS 
>> MESSAGE TO ANYONE. 
>> 
>> -
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
> -- 
> 
> patr...@douglas.co.ukDouglas Instruments Ltd.
> Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
> Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek
> 
> http://www.douglas.co.uk
> Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
> Regd. England 2177994, VAT Reg. GB 480 7371 36 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> --
> 
> * THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM 
> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT 
> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE 
> FROM ADDRESS AND URLs* 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE.

 

Links:
--
[1] https://www.douglas.co.uk/PDB_data.htm




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



To unsubscribe from the CCP4BB list, click the following link:

Re: [ccp4bb] Crystallizing a tough target

[Patrick Shaw Stewart]

Hi Kavya

1. Make a seed stock from the globules or anything else that you think
might be crystalline, and recreen.  In other words, you should add your
seed stock to *random screens* (not optimization experiments).  There could
be many conditions that are in the metastable zone of the phase diagram in
your normal screens - this method can give you crystals in those conditions.

If this works, you'll be in a better position anyway because you'll have
more control - by diluting the seed stock, you can control the number of
crystals per drop.

References:

D'Arcy, A., Villard, F. and Marsh, M., 2007. An automated microseed
matrix-screening method for protein crystallization. *Acta
Crystallographica Section D: Biological Crystallography*, *63*(4),
pp.550-554.

Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock,
P.F., 2011. Random microseeding: a theoretical and practical exploration of
seed stability and seeding techniques for successful protein
crystallization. *Crystal Growth & Design*, *11*(8), pp.3432-3441.


This is how we normally make the seed:

https://www.douglas.co.uk/f_ftp1/rMMS_Procedure.pdf



2. Many years ago, we did some data mining of the crystallization
conditions in a remark in the PDB.  The concentrations that people reported
are below.  There were eight reports where over 100 mg/mL was used.  It
only goes up to 2004.

 https://www.douglas.co.uk/PDB_data.htm




Good luck,

Patrick

[image: image.png]



On Mon, Feb 5, 2024 at 10:27 AM kavyashreem 
wrote:

> Dear All,
>
> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
>
> We have one such candidate, which does not precipitate even at 80mg/ml
> instead forms phase separated globules in crystallization plate, which
> eventually hardens over a period of 1 to 1.5 months (which is florescent
> under UV microscope.)
>
> We tried screening at different pH, but failed to get any hits.
>
> Since we got few conditions in which the phase separated globules
> solidified, we focused on them and expanded with 120mg/ml protein, still
> there were not visible precipitates except for the phase separation. This
> has been a challenging target so far. We have tried with different
> constructs, which unfortunately are not soluble!
>
> Does POMs help in such cases? Or do you have any other suggestion.
>
> Thank you
>
> Regards
>
> Kavya
>
>
>
>
> *CONFIDENTIAL: This email and any files transmitted with it are
> confidential and intended solely for the use of the individual or group to
> whom they are addressed. If you have received this email in error, please
> notify the sender immediately and delete this e-mail from your system. It
> is NOT AUTHORISED to copy, forward, or in any way reveal the contents of
> this message to anyone.*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear Patrick,  

Thank you for the insight! 

Regards 

Kavya 

On 2024-02-06 00:29, Patrick Shaw Stewart wrote:

>> is it logical to say that if the protein is highly charged (either negative 
>> or positive), it is likely to be more soluble and resist crystallization due 
>> to electrostatic repulsion?
> 
> Hi Kavyashreem 
> 
> The project that I mentioned, where we looked at the crystallization 
> conditions reported in the PDB, also looked at the areas of amino acids (and 
> atoms) on the surfaces of proteins and tried to correlate the various groups 
> with crystallization conditions. 
> 
> Positive, negative, charged, polar or hydrophobic groups had very little 
> effect on the concentration of precipitant (we looked at PEG and ammonium 
> sulfate) and seemed not to affect the choice of precipitant (looking at all 
> precipitants). 
> 
> The only thing you could say was that the area of all charged and polar 
> groups as a proportion of the total surface area was roughly constant.  If 
> the charged/polar area was high, the protein was crystallized at slightly 
> higher protein concentrations, and slightly higher concentrations of 
> precipitant were used on average. 
> 
> But it should be possible to make predictions.  DeepMind is probably working 
> on this right now! 
> 
> Best wishes, 
> 
> Patrick 
> 
> On Mon, Feb 5, 2024 at 3:06 PM kavyashreem  wrote: 
> 
> Dear all,  
> 
> Thank you all for your valuable experiences, inputs and references!! I shall 
> try them and hope for some good news! Its good to know there are so many 
> examples of crystallization at such high concentrations.  
> 
> A curious question - is it logical to say that if the protein is highly 
> charged (either negative or positive), it is likely to be more soluble and 
> resist crystallization due to electrostatic repulsion? Our protein has highly 
> positively charged surface, although with some small negative patches.  
> 
> Following are some of the suggestions indicated: 
> 
> 1. Use water (will try) 
> 
> 2. Change buffers, pH temperature - (done) 
> 
> 3. Seeding (will try) 
> 
> 4. Methylating lysines (will try!!) 
> 
> 5. Surface entropy reduction (ongoing) 
> 
> 6. Optimize constructs (done) 
> 
> 7. Different ratios (done) 
> 
> 8. Keep concentrating (will try, the problem is yield is low!) 
> 
> 9. High salt concentrations (will try) 
> 
> 10. Organic solvents (though about it) 
> 
> 11. Mutations (ongoing) 
> 
> Thank you  
> 
> Regards 
> 
> Kavya 
> 
> On 2024-02-05 15:57, kavyashreem wrote: 
> 
> Dear All,  
> 
> Has anyone worked on a protein which is highly soluble even at 80mg/ml? 
> 
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.) 
> 
> We tried screening at different pH, but failed to get any hits. 
> 
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble! 
> 
> Does POMs help in such cases? Or do you have any other suggestion.  
> 
> Thank you  
> 
> Regards 
> 
> Kavya 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> -- 
> * THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM 
> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT 
> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE 
> FROM ADDRESS AND URLs* 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY 

Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear Guillaume,  

That is right, if not for cations it would not have been possible.  

We will check with the surface entropy reduction. Since we are designing
inhibitors for it, we cannot afford to do to much modifications.  

Thank you  

Regards 

Kavya 

On 2024-02-06 03:49, Guillaume Gaullier wrote:

> Hello, 
> 
> Regarding this question: 
> 
>> A curious question - is it logical to say that if the protein is highly 
>> charged (either negative or positive), it is likely to be more soluble and 
>> resist crystallization due to electrostatic repulsion? Our protein has 
>> highly positively charged surface, although with some small negative patches.
> 
> A counter example coming to mind is DNA: it is very strongly charged, yet has 
> been crystallized many times (you can find multiple examples of DNA-only 
> crystal structures in the PDB). Actually charges arranged so regularly along 
> the molecule actually help crystallization, when using an adequate 
> concentration of divalent cations. 
> Since fusion to a crystallizable scaffold protein has been suggested, I would 
> like to add that you could take inspiration from this paper from David 
> Baker's lab to design the scaffold protein (instead of using MBP or similar 
> ones): https://doi.org/10.1038/s41563-023-01683-1 
> Pretty crazy things in there: spontaneous crystallization upon mixing E. coli 
> lysates, crystals that survive being autoclaved... If you know somebody who 
> knows how to use Rosetta for de novo computational design, this might be 
> worth a try. 
> An alternative would be to design de novo a high-affinity and crystallizable 
> binder for your protein. Similarly as the fusion protein approach, de novo 
> design may or may not be easier than alternatives, depending on protein 
> design expertise near you. 
> 
> I hope this helps, 
> 
> Guillaume 
> 
> On 5 Feb 2024, at 22:01, Tao-Hsin Chang  wrote: 
> 
> Dear Kavya,
> 
> I wanted to share with you that we have faced the same issue with a few 
> projects. In addition to the great suggestions given earlier, I recommend 
> trying out fusion proteins, such as the surface entropy reduction MBP mutant, 
> or using protein binding partners like antibodies or natural binders. These 
> strategies can help alter the protein properties and facilitate new crystal 
> contacts. Additionally, it may be worth experimenting with reduced salt 
> concentrations or using a salt-free buffer, akin to using water in place of a 
> buffer.  
> 
> PMID: 32541044 
> PMID: 26850170 
> 
> Wishing you the best of luck, 
> Tao-Hsin Chang 
> 
> On Feb 5, 2024, at 10:05 AM, kavyashreem  wrote: 
> 
> Dear all,  
> 
> Thank you all for your valuable experiences, inputs and references!! I shall 
> try them and hope for some good news! Its good to know there are so many 
> examples of crystallization at such high concentrations.  
> 
> A curious question - is it logical to say that if the protein is highly 
> charged (either negative or positive), it is likely to be more soluble and 
> resist crystallization due to electrostatic repulsion? Our protein has highly 
> positively charged surface, although with some small negative patches.  
> 
> Following are some of the suggestions indicated: 
> 
> 1. Use water (will try) 
> 
> 2. Change buffers, pH temperature - (done) 
> 
> 3. Seeding (will try) 
> 
> 4. Methylating lysines (will try!!) 
> 
> 5. Surface entropy reduction (ongoing) 
> 
> 6. Optimize constructs (done) 
> 
> 7. Different ratios (done) 
> 
> 8. Keep concentrating (will try, the problem is yield is low!) 
> 
> 9. High salt concentrations (will try) 
> 
> 10. Organic solvents (though about it) 
> 
> 11. Mutations (ongoing) 
> 
> Thank you  
> 
> Regards 
> 
> Kavya 
> 
> On 2024-02-05 15:57, kavyashreem wrote: 
> 
> Dear All,  
> 
> Has anyone worked on a protein which is highly soluble even at 80mg/ml? 
> 
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.) 
> 
> We tried screening at different pH, but failed to get any hits. 
> 
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble! 
> 
> Does POMs help in such cases? Or do you have any other suggestion.  
> 
> Thank you  
> 
> Regards 
> 
> Kavya 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, 

Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[Guillaume Gaullier]

Hello,

Regarding this question:

A curious question - is it logical to say that if the protein is highly charged 
(either negative or positive), it is likely to be more soluble and resist 
crystallization due to electrostatic repulsion? Our protein has highly 
positively charged surface, although with some small negative patches.

A counter example coming to mind is DNA: it is very strongly charged, yet has 
been crystallized many times (you can find multiple examples of DNA-only 
crystal structures in the PDB). Actually charges arranged so regularly along 
the molecule actually help crystallization, when using an adequate 
concentration of divalent cations.

Since fusion to a crystallizable scaffold protein has been suggested, I would 
like to add that you could take inspiration from this paper from David Baker’s 
lab to design the scaffold protein (instead of using MBP or similar ones): 
https://doi.org/10.1038/s41563-023-01683-1
Pretty crazy things in there: spontaneous crystallization upon mixing E. coli 
lysates, crystals that survive being autoclaved… If you know somebody who knows 
how to use Rosetta for de novo computational design, this might be worth a try.
An alternative would be to design de novo a high-affinity and crystallizable 
binder for your protein. Similarly as the fusion protein approach, de novo 
design may or may not be easier than alternatives, depending on protein design 
expertise near you.

I hope this helps,

Guillaume


On 5 Feb 2024, at 22:01, Tao-Hsin Chang 
mailto:taohsin.ch...@gmail.com>> wrote:

Dear Kavya,

I wanted to share with you that we have faced the same issue with a few 
projects. In addition to the great suggestions given earlier, I recommend 
trying out fusion proteins, such as the surface entropy reduction MBP mutant, 
or using protein binding partners like antibodies or natural binders. These 
strategies can help alter the protein properties and facilitate new crystal 
contacts. Additionally, it may be worth experimenting with reduced salt 
concentrations or using a salt-free buffer, akin to using water in place of a 
buffer.

PMID: 32541044
PMID: 26850170



Wishing you the best of luck,
Tao-Hsin Chang

On Feb 5, 2024, at 10:05 AM, kavyashreem 
mailto:kavyashr...@instem.res.in>> wrote:


Dear all,

Thank you all for your valuable experiences, inputs and references!! I shall 
try them and hope for some good news! Its good to know there are so many 
examples of crystallization at such high concentrations.

A curious question - is it logical to say that if the protein is highly charged 
(either negative or positive), it is likely to be more soluble and resist 
crystallization due to electrostatic repulsion? Our protein has highly 
positively charged surface, although with some small negative patches.

Following are some of the suggestions indicated:

1. Use water (will try)

2. Change buffers, pH temperature - (done)

3. Seeding (will try)

4. Methylating lysines (will try!!)

5. Surface entropy reduction (ongoing)

6. Optimize constructs (done)

7. Different ratios (done)

8. Keep concentrating (will try, the problem is yield is low!)

9. High salt concentrations (will try)

10. Organic solvents (though about it)

11. Mutations (ongoing)

Thank you

Regards

Kavya

On 2024-02-05 15:57, kavyashreem wrote:

Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



To unsubscribe from the CCP4BB list, click the 

Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[Tao-Hsin Chang]

Dear Kavya,

I wanted to share with you that we have faced the same issue with a few 
projects. In addition to the great suggestions given earlier, I recommend 
trying out fusion proteins, such as the surface entropy reduction MBP mutant, 
or using protein binding partners like antibodies or natural binders. These 
strategies can help alter the protein properties and facilitate new crystal 
contacts. Additionally, it may be worth experimenting with reduced salt 
concentrations or using a salt-free buffer, akin to using water in place of a 
buffer. 

PMID: 32541044
PMID: 26850170
 
Wishing you the best of luck,
Tao-Hsin Chang

> On Feb 5, 2024, at 10:05 AM, kavyashreem  wrote:
> 
> Dear all, 
> 
> Thank you all for your valuable experiences, inputs and references!! I shall 
> try them and hope for some good news! Its good to know there are so many 
> examples of crystallization at such high concentrations. 
> 
> A curious question - is it logical to say that if the protein is highly 
> charged (either negative or positive), it is likely to be more soluble and 
> resist crystallization due to electrostatic repulsion? Our protein has highly 
> positively charged surface, although with some small negative patches. 
> 
> Following are some of the suggestions indicated:
> 
> 1. Use water (will try)
> 
> 2. Change buffers, pH temperature - (done)
> 
> 3. Seeding (will try)
> 
> 4. Methylating lysines (will try!!)
> 
> 5. Surface entropy reduction (ongoing)
> 
> 6. Optimize constructs (done)
> 
> 7. Different ratios (done)
> 
> 8. Keep concentrating (will try, the problem is yield is low!)
> 
> 9. High salt concentrations (will try)
> 
> 10. Organic solvents (though about it)
> 
> 11. Mutations (ongoing)
> 
> Thank you 
> 
> Regards
> 
> Kavya
> 
> On 2024-02-05 15:57, kavyashreem wrote:
> 
>> Dear All, 
>> 
>> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
>> 
>> We have one such candidate, which does not precipitate even at 80mg/ml 
>> instead forms phase separated globules in crystallization plate, which 
>> eventually hardens over a period of 1 to 1.5 months (which is florescent 
>> under UV microscope.)
>> 
>> We tried screening at different pH, but failed to get any hits.
>> 
>> Since we got few conditions in which the phase separated globules 
>> solidified, we focused on them and expanded with 120mg/ml protein, still 
>> there were not visible precipitates except for the phase separation. This 
>> has been a challenging target so far. We have tried with different 
>> constructs, which unfortunately are not soluble!
>> 
>> Does POMs help in such cases? Or do you have any other suggestion. 
>> 
>> Thank you 
>> 
>> Regards
>> 
>> Kavya
>> 
>> 
>> 
>> 
>> 
>> CONFIDENTIAL: This email and any files transmitted with it are confidential 
>> and intended solely for the use of the individual or group to whom they are 
>> addressed. If you have received this email in error, please notify the 
>> sender immediately and delete this e-mail from your system. It is NOT 
>> AUTHORISED to copy, forward, or in any way reveal the contents of this 
>> message to anyone. 
>> 
>> 
>> 
>> 
>> CONFIDENTIAL: This email and any files transmitted with it are confidential 
>> and intended solely for the use of the individual or group to whom they are 
>> addressed. If you have received this email in error, please notify the 
>> sender immediately and delete this e-mail from your system. It is NOT 
>> AUTHORISED to copy, forward, or in any way reveal the contents of this 
>> message to anyone. 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
>> --
>> 
>> 
>> * THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM 
>> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT 
>> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE 
>> FROM ADDRESS AND URLs*
>> 
> 
> 
> 
> 
> 
> CONFIDENTIAL: This email and any files transmitted with it are confidential 
> and intended solely for the use of the individual or group to whom they are 
> addressed. If you have received this email in error, please notify the sender 
> immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
> copy, forward, or in any way reveal the contents of this message to anyone. 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available 

Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[Patrick Shaw Stewart]

>
> is it logical to say that if the protein is highly charged (either
> negative or positive), it is likely to be more soluble and resist
> crystallization due to electrostatic repulsion?


Hi Kavyashreem

The project that I mentioned, where we looked at the crystallization
conditions reported in the PDB, also looked at the areas of amino acids
(and atoms) on the surfaces of proteins and tried to correlate the various
groups with crystallization conditions.

Positive, negative, charged, polar or hydrophobic groups had very little
effect on the concentration of precipitant (we looked at PEG and ammonium
sulfate) and seemed not to affect the choice of precipitant (looking at all
precipitants).

The only thing you could say was that the area of all charged and polar
groups as a proportion of the total surface area was roughly constant.  If
the charged/polar area was high, the protein was crystallized at slightly
higher protein concentrations, and slightly higher concentrations of
precipitant were used on average.

But it should be possible to make predictions.  DeepMind is probably
working on this right now!

Best wishes,

Patrick


On Mon, Feb 5, 2024 at 3:06 PM kavyashreem 
wrote:

> Dear all,
>
> Thank you all for your valuable experiences, inputs and references!! I
> shall try them and hope for some good news! Its good to know there are so
> many examples of crystallization at such high concentrations.
>
> A curious question - is it logical to say that if the protein is highly
> charged (either negative or positive), it is likely to be more soluble and
> resist crystallization due to electrostatic repulsion? Our protein has
> highly positively charged surface, although with some small negative
> patches.
>
> Following are some of the suggestions indicated:
>
> 1. Use water (will try)
>
> 2. Change buffers, pH temperature - (done)
>
> 3. Seeding (will try)
>
> 4. Methylating lysines (will try!!)
>
> 5. Surface entropy reduction (ongoing)
>
> 6. Optimize constructs (done)
>
> 7. Different ratios (done)
>
> 8. Keep concentrating (will try, the problem is yield is low!)
>
> 9. High salt concentrations (will try)
>
> 10. Organic solvents (though about it)
>
> 11. Mutations (ongoing)
>
> Thank you
>
> Regards
>
> Kavya
>
> On 2024-02-05 15:57, kavyashreem wrote:
>
> Dear All,
>
> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
>
> We have one such candidate, which does not precipitate even at 80mg/ml
> instead forms phase separated globules in crystallization plate, which
> eventually hardens over a period of 1 to 1.5 months (which is florescent
> under UV microscope.)
>
> We tried screening at different pH, but failed to get any hits.
>
> Since we got few conditions in which the phase separated globules
> solidified, we focused on them and expanded with 120mg/ml protein, still
> there were not visible precipitates except for the phase separation. This
> has been a challenging target so far. We have tried with different
> constructs, which unfortunately are not soluble!
>
> Does POMs help in such cases? Or do you have any other suggestion.
>
> Thank you
>
> Regards
>
> Kavya
>
>
>
>
> *CONFIDENTIAL: This email and any files transmitted with it are
> confidential and intended solely for the use of the individual or group to
> whom they are addressed. If you have received this email in error, please
> notify the sender immediately and delete this e-mail from your system. It
> is NOT AUTHORISED to copy, forward, or in any way reveal the contents of
> this message to anyone.*
>
>
>
>
> *CONFIDENTIAL: This email and any files transmitted with it are
> confidential and intended solely for the use of the individual or group to
> whom they are addressed. If you have received this email in error, please
> notify the sender immediately and delete this e-mail from your system. It
> is NOT AUTHORISED to copy, forward, or in any way reveal the contents of
> this message to anyone.*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> --
>
> * THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM
> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT
> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE
> FROM ADDRESS AND URLs*
>
>
>
>
>
>
> *CONFIDENTIAL: This email and any files transmitted with it are
> confidential and intended solely for the use of the individual or group to
> whom they are addressed. If you have received this email in error, please
> notify the sender immediately and delete this e-mail from your system. It
> is NOT AUTHORISED to copy, forward, or in any way reveal the contents of
> this message to anyone.*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
 patr...@douglas.co.uk   

Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear all,  

Thank you all for your valuable experiences, inputs and references!! I
shall try them and hope for some good news! Its good to know there are
so many examples of crystallization at such high concentrations.  

A curious question - is it logical to say that if the protein is highly
charged (either negative or positive), it is likely to be more soluble
and resist crystallization due to electrostatic repulsion? Our protein
has highly positively charged surface, although with some small negative
patches.  

Following are some of the suggestions indicated: 

1. Use water (will try) 

2. Change buffers, pH temperature - (done) 

3. Seeding (will try) 

4. Methylating lysines (will try!!) 

5. Surface entropy reduction (ongoing) 

6. Optimize constructs (done) 

7. Different ratios (done) 

8. Keep concentrating (will try, the problem is yield is low!) 

9. High salt concentrations (will try) 

10. Organic solvents (though about it) 

11. Mutations (ongoing) 

Thank you  

Regards 

Kavya 

On 2024-02-05 15:57, kavyashreem wrote:

> Dear All,  
> 
> Has anyone worked on a protein which is highly soluble even at 80mg/ml? 
> 
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.) 
> 
> We tried screening at different pH, but failed to get any hits. 
> 
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble! 
> 
> Does POMs help in such cases? Or do you have any other suggestion.  
> 
> Thank you  
> 
> Regards 
> 
> Kavya 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. 
> 
> CONFIDENTIAL: THIS EMAIL AND ANY FILES TRANSMITTED WITH IT ARE CONFIDENTIAL 
> AND INTENDED SOLELY FOR THE USE OF THE INDIVIDUAL OR GROUP TO WHOM THEY ARE 
> ADDRESSED. IF YOU HAVE RECEIVED THIS EMAIL IN ERROR, PLEASE NOTIFY THE SENDER 
> IMMEDIATELY AND DELETE THIS E-MAIL FROM YOUR SYSTEM. IT IS NOT AUTHORISED TO 
> COPY, FORWARD, OR IN ANY WAY REVEAL THE CONTENTS OF THIS MESSAGE TO ANYONE. 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> -- 
> * THIS IS AN EXTERNAL MAIL AND **DID NOT** ORIGINATE FROM 
> **BLISC(NCBS/INSTEM/CCAMP)** CAMPUS SERVERS. PLEASE VERIFY THE CONTENT 
> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE 
> FROM ADDRESS AND URLs*




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] [External] Re: [ccp4bb] Crystallizing a tough target

[Srivastava, Dhiraj]

While I understand that you want to have protein concentration at its 
solubility limit, i had several proteins which can go to 60-80 mg/ml, they 
seems to get crystallized at 10-15 mg/ml. All you want is saturation  in 
crystallization conditions.
Dhiraj srivastava


From: CCP4 bulletin board  on behalf of Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, February 5, 2024 5:32 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] Crystallizing a tough target

Limited proteolytic might help, too.

https://www.nature.com/articles/nmeth1118


Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile



 Original Message 
On 5 Feb 2024, 10:27, kavyashreem < kavyashr...@instem.res.in> wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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



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



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Oganesyan, Vaheh]

For what it is worth, human serum albumin has been crystallized initially from 
250 mg/ml solution back in ‘90s. When I started working on ternary complex 
hSA-FcRn-Fc (PDB Id 4N0U) was afraid that such high concentration couldn’t be 
achieved with amounts of FcRn I can reasonably express. However, complex got 
crystallized at around 6-8 mg/ml, if I remember it correctly. What I’m trying 
to tell is it might be worth trying to find binding partner for your protein 
and then co-crystallize the complex.

Vaheh Oganesyan, Ph.D.
[cid:image001.png@01DA5817.C3AEDD00]
R | Biologics Engineering
One Medimmune Way, Gaithersburg, MD 20878
T:  301-398-5851
vaheh.oganes...@astrazeneca.com<mailto:oganesy...@medimmune.com>



From: CCP4 bulletin board  On Behalf Of kavyashreem
Sent: Monday, February 5, 2024 5:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallizing a tough target


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Deborah Harrus]

Dear Kavya,

I'm sure there are several similar stories to learn from.

I've worked with a point-mutant of a "normal" protein which made it 
thermostable - as revealed by DSF. It also resulted in an increased 
solubility, and the thing could be concentrated to 100 mg/mL and left on 
the bench for weeks with no alteration. It crystallized, but sadly (and 
understandably) never diffracted.


One option for crystallizing your protein could be to look for buffers 
which make it a bit less soluble? So you wouldn't have to concentrate it 
to so high values, and start nucleation at a lower concentration? Again, 
scanning several buffers/protein concentration ratios, using DSF, would 
be one possibility. You're looking for the sweet spot to start 
nucleation. Don't be afraid to try uncommon buffers, pH values, 
temperatures.


Other option would be to test using nucleanting agent - crystallophores, 
Naomi's nucleant, nanodiamonds, dried seaweed powder, hair... Or even a 
crystal seed from another protein might help.


Good luck!

Deborah

On 05/02/2024 10:27, kavyashreem wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml 
instead forms phase separated globules in crystallization plate, which 
eventually hardens over a period of 1 to 1.5 months (which is 
florescent under UV microscope.)


We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules 
solidified, we focused on them and expanded with 120mg/ml protein, 
still there were not visible precipitates except for the phase 
separation. This has been a challenging target so far. We have tried 
with different constructs, which unfortunately are not soluble!


Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya





*CONFIDENTIAL: This email and any files transmitted with it are 
confidential and intended solely for the use of the individual or 
group to whom they are addressed. If you have received this email in 
error, please notify the sender immediately and delete this e-mail 
from your system. It is NOT AUTHORISED to copy, forward, or in any way 
reveal the contents of this message to anyone.*




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




--
---
Deborah Harrus, Ph.D.
PDBe Archive Project Leader, Biocuration Lead
PDBe - Protein Data Bank in Europe

European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK

http://www.PDBe.org
---



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Roberto Steiner]

HOD crystallised at 150mg/ml
https://www.pnas.org/doi/10.1073/pnas.0909033107

Once I set the wrong time on the centrifuge and it went up to >200 mg/ml. I am 
sure many others have similar stories.

All the best
Roberto


Roberto A Steiner
www.steinerlab.org
https://twitter.com/steiner_lab

roberto.stei...@kcl.ac.uk
Randall Centre for Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
Room 3.10A
New Hunt's House, Guy's Campus
SE1 1UL, London, UK
Phone 0044 20 78488216
Fax0044 20 78486435

roberto.stei...@unipd.it
Dipartimento di Scienze Biomediche
Università degli Studi di Padova
Viale G. Colombo 3
35131 Padova, Italia
Telefono 0039 049 8276409

Responses to emails are not expected outside of your normal working hours.








On 5 Feb 2024, at 11:27, kavyashreem 
mailto:kavyashr...@instem.res.in>> wrote:

You don't often get email from 
kavyashr...@instem.res.in. Learn why this is 
important

Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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




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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[John R Helliwell]

Dear Kavya,
Concanavalin A with glucoside involved 120 mg/ml protein.
Details of the crystallisation are here:-
 https://doi.org/10.1016/0022-2836(87)90198-7 
PDB code 1GIC.
Best wishes,
John 
Emeritus Professor John R Helliwell DSc




> On 5 Feb 2024, at 10:27, kavyashreem  wrote:
> 
> 
> Dear All, 
> 
> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
> 
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.)
> 
> We tried screening at different pH, but failed to get any hits.
> 
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble!
> 
> Does POMs help in such cases? Or do you have any other suggestion. 
> 
> Thank you 
> 
> Regards
> 
> Kavya
> 
> 
> 
> 
> 
> CONFIDENTIAL: This email and any files transmitted with it are confidential 
> and intended solely for the use of the individual or group to whom they are 
> addressed. If you have received this email in error, please notify the sender 
> immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
> copy, forward, or in any way reveal the contents of this message to anyone. 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Jon Cooper]

Limited proteolytic might help, too.

https://www.nature.com/articles/nmeth1118

Best wishes, Jon Cooper. jon.b.coo...@protonmail.com

Sent from Proton Mail mobile

 Original Message 
On 5 Feb 2024, 10:27, kavyashreem wrote:

> Dear All,
>
> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
>
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.)
>
> We tried screening at different pH, but failed to get any hits.
>
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble!
>
> Does POMs help in such cases? Or do you have any other suggestion.
>
> Thank you
>
> Regards
>
> Kavya
>
> CONFIDENTIAL: This email and any files transmitted with it are confidential 
> and intended solely for the use of the individual or group to whom they are 
> addressed. If you have received this email in error, please notify the sender 
> immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
> copy, forward, or in any way reveal the contents of this message to anyone.
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Andre Godoy]

Hi Kavya,

You`r likely working with a very low PI sample, right? . Here are a few 
suggestions
Final purification step at a pH closer to the pI:
 Adjusting the pH during purification to be closer to the pI can reduce the 
solubility and promote precipitation.
Non-PEG precipitants based kits:
Using precipitants like MPD,  AmSO4 , or organic solvents can be effective in 
inducing protein precipitation.
Adding organic solvents (e.g., 4% 1-propanol):
Organic solvents can disrupt the water structure around proteins, leading to 
their precipitation.Make sure to carefully optimize the concentration of the 
organic solvent to avoid denaturation or other adverse effects on the protein.

Glycosylation:
If your protein is glycosylated, this can indeed increasse solubility and 
stability. Consider using enzymes like PNGase F to remove glycosylation if it's 
not crucial for your experiments.

good luck
Andre S. Godoy, PhD 

Universidade de São Paulo
Instituto de Física de São Carlos
Av. João Dagnone, 1100, Jd. Santa Angelina13563-120 - São Carlos, SP, Brazil  

Em segunda-feira, 5 de fevereiro de 2024 às 07:27:28 BRT, kavyashreem 
 escreveu:  
 
 
Dear All, 

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion. 

Thank you 

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.


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



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Tomas Malinauskas]

Hi Kavya,

In addition to other excellent suggestions, I would recommend trying
reductive lysine methylation, which can make the protein more
hydrophobic. PMID 17098187.

Best wishes,
Tomas

On Mon, Feb 5, 2024 at 10:27 AM kavyashreem  wrote:
>
> Dear All,
>
> Has anyone worked on a protein which is highly soluble even at 80mg/ml?
>
> We have one such candidate, which does not precipitate even at 80mg/ml 
> instead forms phase separated globules in crystallization plate, which 
> eventually hardens over a period of 1 to 1.5 months (which is florescent 
> under UV microscope.)
>
> We tried screening at different pH, but failed to get any hits.
>
> Since we got few conditions in which the phase separated globules solidified, 
> we focused on them and expanded with 120mg/ml protein, still there were not 
> visible precipitates except for the phase separation. This has been a 
> challenging target so far. We have tried with different constructs, which 
> unfortunately are not soluble!
>
> Does POMs help in such cases? Or do you have any other suggestion.
>
> Thank you
>
> Regards
>
> Kavya
>
>
>
>
>
> CONFIDENTIAL: This email and any files transmitted with it are confidential 
> and intended solely for the use of the individual or group to whom they are 
> addressed. If you have received this email in error, please notify the sender 
> immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
> copy, forward, or in any way reveal the contents of this message to anyone.
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Gianluca Cioci]

Hi,

I have a small protein that is hugely soluble too.

We could crystallize it in 3.5M ammonium sulfate with the help of a 
ligand that apparently triggered the crystallization.


Try changing the temperature too.

Good luck,

GIA



Le 05/02/2024 à 11:27, kavyashreem a écrit :


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml 
instead forms phase separated globules in crystallization plate, which 
eventually hardens over a period of 1 to 1.5 months (which is 
florescent under UV microscope.)


We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules 
solidified, we focused on them and expanded with 120mg/ml protein, 
still there were not visible precipitates except for the phase 
separation. This has been a challenging target so far. We have tried 
with different constructs, which unfortunately are not soluble!


Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya





*CONFIDENTIAL: This email and any files transmitted with it are 
confidential and intended solely for the use of the individual or 
group to whom they are addressed. If you have received this email in 
error, please notify the sender immediately and delete this e-mail 
from your system. It is NOT AUTHORISED to copy, forward, or in any way 
reveal the contents of this message to anyone.*




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




--
Dr. Gianluca CIOCI
Toulouse Biotechnology Institute (TBI)
https://www.toulouse-biotechnology-institute.fr/en/poles/equipe-cimes/
PICT - Plateforme Intégrée de Criblage de Toulouse
http://www.pict.ipbs.fr/

Tel: +33 (0)5 61 55 97 68
E-mail:ci...@insa-toulouse.fr

TBI - INSA Toulouse
135 avenue de Rangueil
31077 Toulouse CEDEX 04
http://www.toulouse-biotechnology-institute.fr



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Frank von Delft]

Don't forget to try pure water.

On 05/02/2024 10:44, Savvas Savvides wrote:

Dear Kavya,

we encountered this issue for a protein complex at 95 mg/mL involving 
Human Serum Albumin (HSA) and a designed protein binder (Alphabody) as 
described in Pannecoucke et al. 2021 Sci Adv 7 (13), 
DOI:10.1126/sciadv.abe1682.
The breakthrough in that case came from crystallization experiments at 
different temperatures (14C, 21C and 37C), with 37C coming out as the 
clear winner.


best wishes
Savvas


——
Prof. Savvas Savvides
VIB Center for Inflammation Research
Ghent University, Dept. of Biochemistry & Microbiology
Technologiepark 71, 9052 Ghent, Belgium

Email: savvas.savvi...@ugent.be ; savvas.savvi...@irc.vib-ugent.be
Phone: +32 (0)472 928 519 (mobile) ; +32 (0)9 331 36 60 (office)
Web:_https://www.irc.ugent.be/index.php/groups/savvides-unit_


On 5 Feb 2024, at 11:27, kavyashreem  wrote:

Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 
80mg/ml instead forms phase separated globules in crystallization 
plate, which eventually hardens over a period of 1 to 1.5 months 
(which is florescent under UV microscope.)


We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules 
solidified, we focused on them and expanded with 120mg/ml protein, 
still there were not visible precipitates except for the phase 
separation. This has been a challenging target so far. We have tried 
with different constructs, which unfortunately are not soluble!


Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya





*CONFIDENTIAL: This email and any files transmitted with it are 
confidential and intended solely for the use of the individual or 
group to whom they are addressed. If you have received this email in 
error, please notify the sender immediately and delete this e-mail 
from your system. It is NOT AUTHORISED to copy, forward, or in any 
way reveal the contents of this message to anyone.*




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








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








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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] Re [ccp4bb] Crystallizing a tough target

[Andrew Lovering]

Hi Kavya

A few things I would try:

-set up your plates (and incubate) at 4 degrees - perhaps in a cold room
-use Alphafold model to identify patches of charge that you can mutate
-perhaps even introduce a few sticky motifs (e.g. herein and ref 26 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567453/)
-set your drops up at protein:reservoir ratios that aren't 1:1 e.g. 3pro:1res - 
will eventually equilibrate to greater protein concentration

-if super desperate, clone out a small domain, crystallize and then seed back 
the full-length protein with these

Good luck!
Andy



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Savvas Savvides]

Dear Kavya,

we encountered this issue for a protein complex at 95 mg/mL involving Human 
Serum Albumin (HSA) and a designed protein binder (Alphabody) as described in 
Pannecoucke et al. 2021 Sci Adv 7 (13), DOI:10.1126/sciadv.abe1682.
The breakthrough in that case came from crystallization experiments at 
different temperatures (14C, 21C and 37C), with 37C coming out as the clear 
winner.

best wishes
Savvas


——
Prof. Savvas Savvides
VIB Center for Inflammation Research
Ghent University, Dept. of Biochemistry & Microbiology
Technologiepark 71, 9052 Ghent, Belgium

Email: savvas.savvi...@ugent.be ; 
savvas.savvi...@irc.vib-ugent.be
Phone: +32 (0)472 928 519 (mobile) ; +32 (0)9 331 36 60 (office)
Web:https://www.irc.ugent.be/index.php/groups/savvides-unit

On 5 Feb 2024, at 11:27, kavyashreem  wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml instead 
forms phase separated globules in crystallization plate, which eventually 
hardens over a period of 1 to 1.5 months (which is florescent under UV 
microscope.)

We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules solidified, 
we focused on them and expanded with 120mg/ml protein, still there were not 
visible precipitates except for the phase separation. This has been a 
challenging target so far. We have tried with different constructs, which 
unfortunately are not soluble!

Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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




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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Crystallizing a tough target

[Jose A. MARQUEZ]

Dear kavya

There is precedent for this. Check the reference below. This protein was 
crystalized from solution at a concentration of 150 mg/ml. Keep 
concentrating, I guess !


Cell. Volume 98, Issue 4 
, 20 August 
1999, Pages 537-546


https://doi.org/10.1016/S0092-8674(00)81981-9

Best regards

Josan

_
Jose A. Marquez, Senior Scientist
Head of the Crystallization Facility
European Molecular Biology Laboratory, Grenoble.
Delivery address: EMBL, 71, Avenue des Martyrs
38000 Grenoble, France
Postal address: EMBL, 71, Avenue des Martyrs
CS 90181 38042 Grenoble Cedex 9, France
Phone +33 (0)476 20 74 25
Fax. +33 (0)476 20 71 99

https://www.embl.org/groups/marquez/  
https://www.embl.org/services-facilities/grenoble/high-throughput-crystallisation/  
https://htxlab.embl.org/  
_


On 2/5/2024 11:27 AM, kavyashreem wrote:


Dear All,

Has anyone worked on a protein which is highly soluble even at 80mg/ml?

We have one such candidate, which does not precipitate even at 80mg/ml 
instead forms phase separated globules in crystallization plate, which 
eventually hardens over a period of 1 to 1.5 months (which is 
florescent under UV microscope.)


We tried screening at different pH, but failed to get any hits.

Since we got few conditions in which the phase separated globules 
solidified, we focused on them and expanded with 120mg/ml protein, 
still there were not visible precipitates except for the phase 
separation. This has been a challenging target so far. We have tried 
with different constructs, which unfortunately are not soluble!


Does POMs help in such cases? Or do you have any other suggestion.

Thank you

Regards

Kavya





*CONFIDENTIAL: This email and any files transmitted with it are 
confidential and intended solely for the use of the individual or 
group to whom they are addressed. If you have received this email in 
error, please notify the sender immediately and delete this e-mail 
from your system. It is NOT AUTHORISED to copy, forward, or in any way 
reveal the contents of this message to anyone.*




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







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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] Crystallizing a tough target

[kavyashreem]

Dear All,  

Has anyone worked on a protein which is highly soluble even at 80mg/ml? 

We have one such candidate, which does not precipitate even at 80mg/ml
instead forms phase separated globules in crystallization plate, which
eventually hardens over a period of 1 to 1.5 months (which is florescent
under UV microscope.) 

We tried screening at different pH, but failed to get any hits. 

Since we got few conditions in which the phase separated globules
solidified, we focused on them and expanded with 120mg/ml protein, still
there were not visible precipitates except for the phase separation.
This has been a challenging target so far. We have tried with different
constructs, which unfortunately are not soluble! 

Does POMs help in such cases? Or do you have any other suggestion.  

Thank you  

Regards 

Kavya




CONFIDENTIAL: This email and any files transmitted with it are confidential and 
intended solely for the use of the individual or group to whom they are 
addressed. If you have received this email in error, please notify the sender 
immediately and delete this e-mail from your system. It is NOT AUTHORISED to 
copy, forward, or in any way reveal the contents of this message to anyone.



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

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/