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 
> 
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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, 

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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[ccp4bb] Postdoc fellowship for scientists of (mixed) Black heritage at LMB

[Sjors Scheres]

Dear colleagues,

The MRC Laboratory of Molecular Biology (LMB) is proud to launch a new 
three-year fully funded postdoctoral fellowship for scientists from 
Black heritage backgrounds.


The LMB has a proud history of training generations of postdoctoral 
scientists who have gone on to great things across the biomedical 
sciences and beyond. We want to enable scientists of Black heritage who 
are brimming with potential and passion for molecular biology and the 
biomedical sciences to learn and grow their experience in our unique 
collaborative research environment based in our state-of-the-art 
building on the Cambridge Biomedical Campus. The LMB will provide 
support for Fellows to move their career forward including a generous 
training budget and mentoring from LMB scientists.


While the paucity of Black scientists is evident across the UK’s higher 
education sector, the LMB is committed to playing its part to improve 
the representation of Black heritage scientists and to provide role 
models for future generations of scientists. This is why we are offering 
targeted funding for two Fellows every year.


This scheme is open to people who self-identify as being from a Black 
heritage background, including a mixed Black heritage background. 
Applicants must be either: UK domiciled researchers who wish to conduct 
their research in the UK, or non-UK domiciled researchers who have 
studied in the UK for a degree and/or PhD.


Please see our website for more information: 
https://www2.mrc-lmb.cam.ac.uk/recruitment/rising-talent-fellowship/


Best wishes,

Sjors



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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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> 
> 
> 
> 
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[ccp4bb] Post Doc position at QMUL

[Aravindan Ilangovan]

Dear All

We are looking for a post-doctoral research associate with expertise in 
cryo-EM/cryo-ET to join our research group in the Centre for Molecular Cell 
Biology at Queen Mary University of London. This Wellcome Trust funded post 
will work towards understanding the molecular mechanism of DNA replication 
initiation in bacteria. This structural biology project will take advantage of 
the expertise in the lab working with nucleoprotein complexes aided by 
dedicated cryo-EM facility. The group has routine access to the Titan Krios 
microscopes at LonCEM and eBIC and an in-house computing infrastructure for 
data processing.


Details about the position and a link to the application system can be found 
here



https://www.qmul.ac.uk/jobs/vacancies/items/9305.html



Feel free to contact me (a.ilango...@qmul.ac.uk) 
if you have any specific queries about this post.



Best

Aravindan

Aravindan Ilangovan
Abernethy building | QMUL
Whitechapel
London | E1 2AT

www.ilangovan-lab.com/





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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
> is NOT AUTHORISED to copy, forward, or in any way reveal the contents of
> this message to anyone.*
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> --
>
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> CAREFULLY. PLEASE DO NOT SUBMIT USER CREDNTIALS ON ANY FORMS AND VERIFY THE
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>
>
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>
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 patr...@douglas.co.uk   

[ccp4bb] Postdoctoral position University of Oxford (UK)

[Abimael Cruz Migoni]

Title:  Postdoctoral position University of Oxford (UK)

Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, 
Headington, Oxford OX3 9DU

About the role

Working with Prof. Peter McHugh's research group to contribute to the study of 
DNA repair.

The post holder will be responsible for characterising repair factors through 
structural biology techniques, with a focus on cryo-electron microscopy. Ideal 
candidates should have expertise in molecular cloning, protein expression, 
purification, and structural methods, preferably in crystallography and 
electron microscopy. Knowledge of DNA modification enzymes and a background in 
DNA damage and repair fields are highly desirable.
Your responsibilities encompass testing hypotheses, managing academic research, 
developing scientific techniques, characterizing nucleases, staying updated on 
DNA repair advancements, and contributing to projects and grant proposals. 
You'll attend meetings, present reports, engage in scientific presentations, 
write for publication, and ensure compliance with safety protocols. 
Additionally, you'll manage lab equipment, provide guidance to team members, 
train juniors, and participate in public engagement activities.

The post will be based at the Weatherall Institute of Molecular Medicine, John 
Radcliffe Hospital, Headington, Oxford OX3 9DU , and is offered on a full-time 
basis, fixed-term for 2 years in first instance.

Please apply via this link:


https://my.corehr.com/pls/uoxrecruit/erq_jobspec_version_4.display_form?p_company=10_internal_external=E_display_in_irish=N_process_type=_applicant_no=_form_profile_detail=_display_apply_ind=Y_refresh_search=Y_recruitment_id=170882


--

Dr. Abimael Cruz Migoni
Postdoctoral Scientist

Prof. Peter McHugh lab
Weatherall Institute of Molecular Medicine,
Dept. of Oncology
University of Oxford
John Radcliffe Hospital/Headley Way,
Oxford OX3 9DS



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[ccp4bb] postdoctoral position - time-resolved serial crystallography

[Przemyslaw Nogly]

Dear All,

We have an exciting opening for a postdoctoral position in my group at the 
Jagiellonian University to investigate photoreceptor proteins with 
time-resolved serial crystallography.

For details and applications:
https://www.nature.com/naturecareers/job/12813689/postdoctoral-researcher-at-the-dioscuri-centre-for-structural-dynamics-of-receptors/?LinkSource=PremiumListing
Closing date is 17.03.2024

Best regards,
Przemek

Przemyslaw Nogly, Ph.D.
Group Leader of the Dioscuri Centre for Structural Dynamics of Receptors
Faculty of Biochemistry, Biophysics and Biotechnology
Jagiellonian University
Gronostajowa 7, Room C041
30-387 Krakow, Poland
+48 12 664 6339 
https://noglylab.eu/



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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 
> 
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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




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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



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




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intended solely for the use of the individual or group to whom they are 
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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





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confidential and intended solely for the use of the individual or 
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--
---
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
---



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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




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intended solely for the use of the individual or group to whom they are 
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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. 
> 
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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.
>
> ---
>
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Re: [ccp4bb] Error in "Prepare and validate files for deposition" task"

[Deborah Harrus]

Dear Maria,

Apologies for late reply. I'm not sure which version of 
"adding_stats_to_mmcif" is in CCP4 at the moment, but if you're facing 
the issue again in the future please could you try using the latest 
version at https://github.com/wwPDB/py-adding_stats_to_mmcif ? If it 
doesn't work, please feel free to send me your file and I will troubleshoot.


Kind regards,

Deborah Harrus

PDBe

On 30/01/2024 17:36, mdimarog wrote:

Dear all,

I am trying to deposit 2 structures to the PDBe, solved and refined in 
CCP4 (version 7), and checked in PRIVATEER as they have a lot of 
sugars in. To this end, I used "Prepare and validate files for 
deposition" task, as I had previously (and succesfully) done. However, 
and in spite of switching to the latest CCP4 version (8.0.017), I keep 
getting this error message "adding_stats_to_mmcif_i2_gui:Error in 
wrapper Failed to add 204". I am using macOS Monterey, version 12.6.5.


Does anybody have an idea what is going wrong? If not, does anybody 
know a different way to submit the reflection data to the PDBe? For 
example, would this be somehow possible just using the output from 
Refmac and AIMLESS? I wouldn't want to repeat the 
scaling/MR/refinement procedure because there are a lot of sugars 
there, quite difficult to built.


Many thanks in advance for your help!

Kind regards,

Maria





--
---
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
---



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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




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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
>
>
>
>
>
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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





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Toulouse Biotechnology Institute (TBI)
https://www.toulouse-biotechnology-institute.fr/en/poles/equipe-cimes/
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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





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[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



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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




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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





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confidential and intended solely for the use of the individual or 
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[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




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[ccp4bb] Diffraction Methods 2024

[Winter, Graeme (DLSLtd,RAL,LSCI)]

Dear CCP4BB,

As some of you may remember we are keeping the spirit of the GRC in diffraction 
methods in structural biology alive. Accordingly I would like to announce, on 
behalf of the meeting organisers

Diffraction Methods 2024

Which will be held 22nd - 27th July 2024 at the Harnack House in Berlin, with 
the focus around the tension between experiments and measurements in structural 
biology, specifically around diffraction methods.

For more detail about the meeting and registration please visit

https://events.gwdg.de/event/650/

We are hoping to encourage as many early career researchers as possible - the 
last GRC had a fantastic mix of the new and the experienced which we would like 
to replicate. Therefore if you consider yourself to be early career person, 
please consider attending!

We look forward to seeing you there - if you have any questions please get in 
touch via diffmet2...@physnet.uni-hamburg.de

Best wishes Graeme

On behalf of the organisers of both events, Graeme Winter, Kunio Hirata, Helena 
Taberman, Ali Ebrahim, Arwen Pearson and Ashwin Chari

[https://events.gwdg.de/event/650/attachments/533/901/Diff_Met2024_Poster.001.jpeg]


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