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 
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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, FORWARD

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




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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. 
>> 
>> 
>> 
>> 
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>> addressed. If you have received this email in error, please notify the 
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>> 
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>> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE 
>> FROM ADDRESS AND URLs*
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> 
> 
> 
> 
> 
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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
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-- 
 patr...@douglas.co.u

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 
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