Re: [ccp4bb] Co-crystallization and thermal shift assay

[Saif Mohd]

Thank you everyone for the detailed explanations/suggestions and sharing
your successful experience. It is very helpful.

Earlier I had thought that maybe with  ΔTm, I could select the most
promising molecules. But now it is unlikely the case.
I should have also mentioned that the IC50 values for all the compounds are
between 1-5uM.

Thanks again,
Have a wonderful weekend,
Saif


On Thu, Feb 25, 2021 at 7:38 PM Maria Cristina Nonato 
wrote:

> *Dear Saif*
>
> *Hope you are doing well and safe!*
>
> 1) How much change in Tm (ΔTm) in a thermal shift assay is considered to
> be significant ?
>
> *As it has already been mentioned there is no specific cutoff  for deltaTm
> to be considered significant. DeltaTm depends on many factors, including
> the type of dye you use, protein structure and where your compound binds*.
>
>
> 2) A negative  ΔTm infers that the compound is making the protein
> unstable. In such a case, will the co-crystallization be difficult or just
> impossible or on the contrary it shouldn't matter much?
>
>
> *A negative detaTm means the compound is binding to a different
> conformational state of the protein, compared to the native one. I would
> not consider co-crystallization more difficult or impossible in the
> presence of those compounds, but I would definitely screen for different
> crystallization conditions.*
>
> *Good luck*
>
> *Cristy*
> *#womeninscience*
>
>
> Em qui., 25 de fev. de 2021 às 11:56, Saif Mohd 
> escreveu:
>
>> Hello everyone,
>>
>> 1) How much change in Tm (ΔTm) in a thermal shift assay is considered to
>> be significant ?
>>
>> 2) A negative  ΔTm infers that the compound is making the protein
>> unstable. In such a case, will the co-crystallization be difficult or just
>> impossible or on the contrary it shouldn't matter much?
>>
>>
>> Thanks and best regards,
>> Saif
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
>



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Re: [ccp4bb] Co-crystallization and thermal shift assay

[Maria Cristina Nonato]

*Dear Saif*

*Hope you are doing well and safe!*

1) How much change in Tm (ΔTm) in a thermal shift assay is considered to be
significant ?

*As it has already been mentioned there is no specific cutoff  for deltaTm
to be considered significant. DeltaTm depends on many factors, including
the type of dye you use, protein structure and where your compound binds*.

2) A negative  ΔTm infers that the compound is making the protein unstable.
In such a case, will the co-crystallization be difficult or just impossible
or on the contrary it shouldn't matter much?


*A negative detaTm means the compound is binding to a different
conformational state of the protein, compared to the native one. I would
not consider co-crystallization more difficult or impossible in the
presence of those compounds, but I would definitely screen for different
crystallization conditions.*

*Good luck*

*Cristy*
*#womeninscience*


Em qui., 25 de fev. de 2021 às 11:56, Saif Mohd 
escreveu:

> Hello everyone,
>
> 1) How much change in Tm (ΔTm) in a thermal shift assay is considered to
> be significant ?
>
> 2) A negative  ΔTm infers that the compound is making the protein
> unstable. In such a case, will the co-crystallization be difficult or just
> impossible or on the contrary it shouldn't matter much?
>
>
> Thanks and best regards,
> Saif
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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Re: [ccp4bb] Co-crystallization and thermal shift assay

[Prem Prakash]

Hi Saif,

If your goal is to perform co-crystallization, I am completely agreed with
David's suggestions. Delta Tm does matter in the co-crystallization but
that's not always the case.  I have some experience with protein complexes
where I successfully co-crystallized by using really high molar ratio of
substrate (1: 20, 1: 50, and even 1:200 given your substrate is not too
much expensive). Try some additives relevant to your system.

Good luck
Prem

On Thu, 25 Feb 2021, 08:56 Saif Mohd,  wrote:

> Hello everyone,
>
> 1) How much change in Tm (ΔTm) in a thermal shift assay is considered to
> be significant ?
>
> 2) A negative  ΔTm infers that the compound is making the protein
> unstable. In such a case, will the co-crystallization be difficult or just
> impossible or on the contrary it shouldn't matter much?
>
>
> Thanks and best regards,
> Saif
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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[ccp4bb] Fwd: [ccp4bb] Co-crystallization and thermal shift assay

[mesters]

Hello,

we had an interesting case in the lab many years ago... Using the 
shift-assay, a student managed to identify conditions that markedly 
stabilized the protein of interest. To cut a long story short, in the 
end it turned out conditions were identified that gave monomers while 
the biological active unit is clearly multimeric! Actually, the monomer 
never crystallized while the multimer did...


Keep in mind that a compound may induce a structural change and that the 
resulting structure may indeed be less stable. An increase in ΔTm would 
certainly "desirable" but this alone is not to be used as a measure. If 
you are interested in the complex, a more important parameter will be 
the affinity of the compound for the protein. Example, let's assume the 
affinity is about 50 µM, then you will need at least 10 times that value 
in the crystallization droplet to achieve about 90% occupation. Problem 
may be with concentrations > 1 mM, you can not achieve that high 
concentration in the solute and will have to add for example DMSO which 
may in turn have negative efefcts on your protein of interest. 
Furthermore, strange scenarios could be, the compound itself interferes 
in protein-protein contact formation at higher concentrations or, could 
promote protein-protein contact formation (work as a glue) and not show 
up in the active site at all...


Question in the end will be not about ΔTm (which is one useful tool for 
identifying buffers or compounds worth using/testing), but whether the 
complex can in fact be co-crystallized (the ultimate "test"!).  So yes, 
go ahead and test it.


Best,

Jeroen

--
*Dr.math. et dis. nat.Jeroen R. Mesters*
Deputy, Lecturer, Program Coordinator /Infection Biology
/ 
Visiting 
Professorship (South Bohemian University) in Biophysics

*University of Lübeck*
Center for Structural and Cell Biology in Medicine
*Institute of Biochemistry*

Tel +49 451 3101 3105 (secretariate 3101)
Fax +49 451 3101 3104
jeroen.mest...@uni-luebeck.de 
www.biochem.uni-luebeck.de 

*Ratzeburger Allee 160
23538 Lübeck, Schleswig-Holstein
Germany*

Am 25.02.21 um 15:55 schrieb Saif Mohd:

Hello everyone,

1) How much change in Tm (ΔTm) in a thermal shift assay is considered 
to be significant ?


2) A negative  ΔTm infers that the compound is making the protein 
unstable. In such a case, will the co-crystallization be difficult or 
just impossible or on the contrary it shouldn't matter much?



Thanks and best regards,
Saif




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Re: [ccp4bb] Co-crystallization and thermal shift assay

[David Briggs]

Hi Saif,

Whilst in very general terms, ∆Tm does correlate with binding affinity (there 
will of course always be exceptions to this rule), I don't think there is a 
cutoff beyond which you know that co-crystallisation is feasible. The degree of 
stabilisation will depend very much on the system you are studying.

I've had proteins crystallise with ligands with a very modest ∆Tms (1-2ºC) and 
then failed to get the same protein to crystallise with ligands that give a 
15-20ºC ∆Tm.

I certainly wouldn't let a TSA result dissuade me from trying to co-crystallise 
a protein with a ligand if the hoped-for structure was important for answering 
whatever biological question I'm asking.

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Saif Mohd 

Sent: 25 February 2021 14:55
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Co-crystallization and thermal shift assay

Hello everyone,

1) How much change in Tm (ΔTm) in a thermal shift assay is considered to be 
significant ?

2) A negative  ΔTm infers that the compound is making the protein unstable. In 
such a case, will the co-crystallization be difficult or just impossible or on 
the contrary it shouldn't matter much?


Thanks and best regards,
Saif




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[ccp4bb] Co-crystallization and thermal shift assay

[Saif Mohd]

Hello everyone,

1) How much change in Tm (ΔTm) in a thermal shift assay is considered to be
significant ?

2) A negative  ΔTm infers that the compound is making the protein unstable.
In such a case, will the co-crystallization be difficult or just impossible
or on the contrary it shouldn't matter much?


Thanks and best regards,
Saif



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