[ccp4bb] co-crystallization

[Yvonne TAN Yih Wan]

Hi ,

I am co-crystallizing a protein with compound and would like to know how much 
of compound to add to protein solution to start with. I know that the protein 
binds compound in a 1 to 1 ratio but also noticed that the compound 
precipitates out of solution when DMSO is diluted off. Where should I start of? 
A 1 protein :2 compound ratio or more? And what is the best method to determine 
if the binding is homogeneous (that all protein has got a compound in it)?

Any suggestions would help. Thanks

TY

[ccp4bb] Co-crystallization

[Priya Mudgal]

Dear All,
I am trying to co-crystallize a protein with substrate/substrate analogue
and for some reason it is not working.  I have tried to co-crystallize with
upto 2 mM substrate and it does not bind.  The protein precipitates out if I
go higher concentration.  Soaking does not work either.  Is there any
suggestion that I should try before I give up on this.

Thanks a lot.
Priya

-- 
Priya Mudgal
PhD Student
Duke University

[ccp4bb] co-crystallization

[yangliuqing]

Hello,everyone,
I have a question for cocrystallization, is there some relationship between Km 
value and substrate concentration when making cocrystallization? How can I know 
the substrate is enough for binding?
Thank you very much!
liuqing
_
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Re: [ccp4bb] co-crystallization

[Shilong Fan]

for me, I prefer to sock these compounds into your crystal. it will much more 
easy than co-crystallizaiton. But each protein should be different.

Normally when I star to co-crystallization with small compound, I will set up 
the complex with 1:1.2 molar ratio as first trial to see what should happen.

As DMESO, you can't get rid of them. But you can find  as much lower 
concentraiton as you can.  

Re: [ccp4bb] co-crystallization

[Annie Hassell]

Hi YT-

We normally prepare our ligand stocks in DMSO and add this to the protein in 
3-fold  molar excess.  The majority of our ligands are quite insoluble and 
precipitate when the DMSO concentration decreases upon addition to the 
protein... so I am not surprised that you are seeing this.  If your 
compound does not bind your protein tightly, you might consider using a 5-fold 
molar excess of ligand.

Some proteins crash out if the protein concentration in high when you add the 
ligand.  For those situations, we complex the ligand with dilute protein (1-2 
mg/ml), and then concentrate this for crystallization trials.  I have had 
proteins where we had to complex the dilute protein with ligand, and then let 
it sit overnight at 4C before we concentrated the protein.  We normally 
incubate the protein+ligand at 4C for 1-3 hours for binding before we set up 
the crystallization experiments.

Another scenario might be addition of ligand to the protein followed by 
incubation at room temp for ~1hr.  Then centrifuge at 4C, keep protein at 4C 
and set up your trays.

Hope this helps!
annie







From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne 
TAN Yih Wan
Sent: Thursday, August 25, 2011 2:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] co-crystallization

Hi ,

I am co-crystallizing a protein with compound and would like to know how much 
of compound to add to protein solution to start with. I know that the protein 
binds compound in a 1 to 1 ratio but also noticed that the compound 
precipitates out of solution when DMSO is diluted off. Where should I start of? 
A 1 protein :2 compound ratio or more? And what is the best method to determine 
if the binding is homogeneous (that all protein has got a compound in it)?

Any suggestions would help. Thanks

TY

Re: [ccp4bb] co-crystallization

[Prince, D Bryan]

Dear TY,



Typically between 5-10x molar concentration over the protein is enough to 
ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM 
to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO 
NOT REMOVE it. I learned to my chagrin that you change all the dynamics of the 
drop when you do. I ended up with empty crystals until I left the precipitate 
in place. Think of it this way-



Free protein + compound ↔ protein:compound complex + precipitate (mix of 
protein + compound)



If you change the equilibrium by removing the precipitate, you remove the 
“pressure” on the P:C complex, and it will dissociate to P + C. The precipitate 
acts as a reserve of protein and compound, thus favoring (or stabilizing) the 
P:C complex. I set drops up as a slurry frequently, and if I get crystals, they 
always have the compound bound. Pay attention to the drops if you are 
screening, because it will be important to note what makes the precipitated 
solution better (clear drops=solubilizing) or worse (aggregated drops=decreased 
solubility of your complex). You can also try suspending your compound in LMW 
PEG’s (200-400 FW) instead of DMSO. Either way, try using DMSO (~20%) or LMW 
PEG (~30%) depending on your crystallization conditions as a cryoprotectant. 
Any crystals that grow have some small amount of those agents in them already, 
so they should be more tolerant of them in higher concentrations.



Best of luck!



Bryan



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne 
TAN Yih Wan
Sent: Thursday, August 25, 2011 2:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] co-crystallization



Hi ,



I am co-crystallizing a protein with compound and would like to know how much 
of compound to add to protein solution to start with. I know that the protein 
binds compound in a 1 to 1 ratio but also noticed that the compound 
precipitates out of solution when DMSO is diluted off. Where should I start of? 
A 1 protein :2 compound ratio or more? And what is the best method to determine 
if the binding is homogeneous (that all protein has got a compound in it)?



Any suggestions would help. Thanks



TY


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Re: [ccp4bb] Co-crystallization

[Kontopidis George]

What is the solubility of substrate?

Try analogues with higher solubility. The 2mM may not be 2mM in the
solution! 

The fact that you added there does not mean that it goes to the solution.

If solubility is indeed high and Ki/[solubility]>1 you should be able to see
some density in the binding site.

If not then check the following

is binding site accessible in the crystallise protein?

If binding site is blocked in the crystal lattice substrate cannot go there.

The crystallisation conditions you try to crystallise protein with substrate
are different from the native (unliganded)?

May be you have to try different conditions

George 

  _  

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Priya
Mudgal
Sent: Thursday, November 27, 2008 11:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Co-crystallization

 

Dear All,
I am trying to co-crystallize a protein with substrate/substrate analogue
and for some reason it is not working.  I have tried to co-crystallize with
upto 2 mM substrate and it does not bind.  The protein precipitates out if I
go higher concentration.  Soaking does not work either.  Is there any
suggestion that I should try before I give up on this.

Thanks a lot.
Priya

-- 
Priya Mudgal
PhD Student
Duke University

Re: [ccp4bb] Co-crystallization

[Joern Krausze]

Dear Priya,

How long did you soak your crystals? 

I had a similar problem. When I tried to co-crystallize my protein with 
its substrate (5 mM) I never got any substrate bound. I tried soaking 
with higher concentration (up to 50 mM) and this didn't work either. The 
crystals were infinitely stable in the soaking solution but even after 72 
hours there was no substrate bound. When I was almost giving up I tried 
one last crystal which was in the soaking solution for a long time. I mean 
a very long, several weeks or so. In this crystal the substrate was bound 
by the protein. I don't really know why the binding kinetics was so slow. 
The enzyme's affinity for the substrate was quite high and I didn't expect 
an unusually long soaking time.

Maybe a very long soaking time could also help in your case?

Joern
 



From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of 
Priya Mudgal
Sent: Thursday, November 27, 2008 11:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Co-crystallization

 

Dear All,
I am trying to co-crystallize a protein with substrate/substrate analogue 
and for some reason it is not working.  I have tried to co-crystallize
with upto 2 mM substrate and it does not bind.  The protein precipitates 
out if I go higher concentration.  Soaking does not work either.  Is
there any suggestion that I should try before I give up on this.

Thanks a lot.
Priya

--
Priya Mudgal
PhD Student
Duke University


**
Address:
Joern Krausze
University of Leipzig
Centre for Biotechnology and Biomedicine
Deutscher Platz 5
04103 Leipzig
Germany

eMail:  [EMAIL PROTECTED]
Phone:  +49 (0)341 9731312
Fax:+49 (0)341 9731319
**

Re: [ccp4bb] Co-crystallization

[Kontopidis George]

Very long soaking time will also help.
In your question why the binding kinetics was so slow
Here are some explanations
Diffusion Rate depends from solution viscosity, size of the molecule that
moves in this solution and the pores it has to go through. 
The binding kinetics in the crystals are usually low (compare to solution) 
Because 
-pores 
ligand has to diffuse through solvent channels that sometimes are really
narrow (1 molecule only can go through) 
-size
Ligands sometime are big molecules (ions are moving really fast)
-viscosity
the worst of all the experiment (most of the times) take place in a very
viscous environment  (high PEG, high salt concentration)

George

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Joern
Krausze
Sent: Friday, November 28, 2008 4:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Co-crystallization

Dear Priya,

How long did you soak your crystals? 

I had a similar problem. When I tried to co-crystallize my protein with 
its substrate (5 mM) I never got any substrate bound. I tried soaking 
with higher concentration (up to 50 mM) and this didn't work either. The 
crystals were infinitely stable in the soaking solution but even after 72 
hours there was no substrate bound. When I was almost giving up I tried 
one last crystal which was in the soaking solution for a long time. I mean 
a very long, several weeks or so. In this crystal the substrate was bound 
by the protein. I don't really know why the binding kinetics was so slow. 
The enzyme's affinity for the substrate was quite high and I didn't expect 
an unusually long soaking time.

Maybe a very long soaking time could also help in your case?

Joern
 



From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of 
Priya Mudgal
Sent: Thursday, November 27, 2008 11:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Co-crystallization

 

Dear All,
I am trying to co-crystallize a protein with substrate/substrate analogue 
and for some reason it is not working.  I have tried to co-crystallize
with upto 2 mM substrate and it does not bind.  The protein precipitates 
out if I go higher concentration.  Soaking does not work either.  Is
there any suggestion that I should try before I give up on this.

Thanks a lot.
Priya

--
Priya Mudgal
PhD Student
Duke University


**
Address:
Joern Krausze
University of Leipzig
Centre for Biotechnology and Biomedicine
Deutscher Platz 5
04103 Leipzig
Germany

eMail:  [EMAIL PROTECTED]
Phone:  +49 (0)341 9731312
Fax:+49 (0)341 9731319
**

Re: [ccp4bb] co-crystallization

[Juergen Bosch]

The Km changes with your reservoir, so predictions are limited. In  
general if you have a low Km this is favourable but not a given that  
your ligand will be found in the electron density map. As a starting  
point try a molar ratio of >3 of the ligand to your protein and you  
can go as high as you want (assuming it's soluble and does not cost  
you an arm and a leg). 50 fold excess has been used in some cases. And  
remember for your cryo-step to include your ligand in sufficient  
amounts !


Jürgen

On 1 Dec 2008, at 05:31, yangliuqing wrote:


Hello,everyone,
I have a question for cocrystallization, is there some relationship  
between Km value and substrate concentration when making  
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing

八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！


-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone:   +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

Re: [ccp4bb] co-crystallization

[mesters]

Yes!, there is:

the fraction of occupied protein with substance can be calculated: S / 
(S + Km) with S being the concentration of the compound.


So, if S = Km, half of the sites are occupied (it follows from 
Michaelis-Menten theory).


In order to saturate the enzyme for 90,90909 % with the compound:

1) S = 10 x Kd (concentration of S at least 10 times the Kd)
and
2) S > P (total concentration of S must be larger than total 
concentration of protein or "binding sites")


Depending on the solubility of the compound, this is not always 
possible. In such a case, you need to use DMSO and/or add solid compound 
to the protein solution and leave it for quite some time for the 
compound to finally bind to the protein.


- J. -


yangliuqing wrote:

Hello,everyone,
I have a question for cocrystallization, is there some relationship 
between Km value and substrate concentration when making 
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 




--
Dr. Jeroen R. Mesters
Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
Institut für Biochemie, Universität zu Lübeck
Zentrum für Medizinische Struktur- und Zellbiologie
Ratzeburger Allee 160, D-23538 Lübeck
Tel: +49-451-5004065, Fax: +49-451-5004068
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.selfish-brain.org
Http://www.opticryst.org
--
If you can look into the seeds of time and say
which grain will grow and which will not - speak then to me  (Macbeth)
--

Re: [ccp4bb] co-crystallization

[Engin Ozkan]

As said, your Km is different in mother liquor than in your reaction 
conditions, but even that is not the end of it: You ligand/substrate might be 
inducing the slightest of all conformations in your protein that interferes 
with crystallization, or might block crystal contacts.  Then, you can either 
(1) get no crystals any longer, or (2) get no substrate in your crystals, 
depending on whichever wins energetically, substrate binding or crystal 
formation.  Therefore, being orders of magnitude above physiological Km/Kd is 
still not a guarantee for getting crystals of a complex. So, dwelling 
obsessively on molar ratios and scratching your head becomes irrelevant after a 
while, one should just do the experiment with a good molar ratio as suggested 
in this thread, and accept the verdict of the gods of crystallization.

Interestingly enough, your substrate-protein complex might crystallize more 
efficiently, or crystallize in different conditions, so not all is negative.

Engin

P.S. You are talking about Km and a substrate, but I hope you have a substrate 
analog, inactive enzyme or you are trying to capture the product complex, 
otherwise you might be in for a surprise when you solve the structure.

- Original Message -
From: "Juergen Bosch" <[EMAIL PROTECTED]>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, December 1, 2008 6:35:24 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] co-crystallization

The Km changes with your reservoir, so predictions are limited. In general if 
you have a low Km this is favourable but not a given that your ligand will be 
found in the electron density map. As a starting point try a molar ratio of >3 
of the ligand to your protein and you can go as high as you want (assuming it's 
soluble and does not cost you an arm and a leg). 50 fold excess has been used 
in some cases. And remember for your cryo-step to include your ligand in 
sufficient amounts ! 


Jürgen 



On 1 Dec 2008, at 05:31, yangliuqing wrote: 



Hello,everyone, 
I have a question for cocrystallization, is there some relationship between Km 
value and substrate concentration when making cocrystallization? How can I know 
the substrate is enough for binding? 
Thank you very much! 
liuqing 


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 






- 
Jürgen Bosch 
University of Washington 
Dept. of Biochemistry, K-426 
1705 NE Pacific Street 
Seattle, WA 98195 
Box 357742 
Phone: +1-206-616-4510 
FAX: +1-206-685-7002 
Web: http://faculty.washington.edu/jbosch 

Re: [ccp4bb] co-crystallization

[Edward A. Berry]

mesters wrote:

Yes!, there is:

the fraction of occupied protein with substance can be calculated: S / 
(S + Km) with S being the concentration of the compound.


So, if S = Km, half of the sites are occupied (it follows from 
Michaelis-Menten theory).


But- one warning (perhaps obvious but I think worth pointing out):
S here is the _free_ concentration. The total concentration you have
to add must include the bound ligand also. To achieve 50% saturation
you need to add 50% of the protein concentration plus 1 x Km.

In enzyme assays, where the enzyme may be picomolar, the amount
bound is often insignificant. But in crystallization experiments
the protein is often hundreds of micromolar, and Ki's < 1 uM,
the Ki becomes irrelevant- just add stoichiometric ligand and
a little extra.
Ed



In order to saturate the enzyme for 90,90909 % with the compound:

1) S = 10 x Kd (concentration of S at least 10 times the Kd)
and
2) S > P (total concentration of S must be larger than total 
concentration of protein or "binding sites")


Depending on the solubility of the compound, this is not always 
possible. In such a case, you need to use DMSO and/or add solid compound 
to the protein solution and leave it for quite some time for the 
compound to finally bind to the protein.


- J. -


yangliuqing wrote:

Hello,everyone,
I have a question for cocrystallization, is there some relationship 
between Km value and substrate concentration when making 
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 

Re: [ccp4bb] co-crystallization

[Phoebe Rice]

Just to be a pedantic pain - Km is not necessarily Kd.  I
think that assumption only holds if the chemical step
following substrate binding is rate-limiting.
   Phoebe


 Original message 
>Date: Mon, 1 Dec 2008 15:34:59 +0100
>From: mesters <[EMAIL PROTECTED]>  
>Subject: Re: [ccp4bb] co-crystallization  
>To: CCP4BB@JISCMAIL.AC.UK
>
>Yes!, there is:
>
>the fraction of occupied protein with substance can be
calculated: S / 
>(S + Km) with S being the concentration of the compound.
>
>So, if S = Km, half of the sites are occupied (it follows from 
>Michaelis-Menten theory).
>
>In order to saturate the enzyme for 90,90909 % with the compound:
>
>1) S = 10 x Kd (concentration of S at least 10 times the Kd)
>and
>2) S > P (total concentration of S must be larger than total 
>concentration of protein or "binding sites")
>
>Depending on the solubility of the compound, this is not always 
>possible. In such a case, you need to use DMSO and/or add
solid compound 
>to the protein solution and leave it for quite some time for the 
>compound to finally bind to the protein.
>
>- J. -
>
>
>yangliuqing wrote:
>> Hello,everyone,
>> I have a question for cocrystallization, is there some
relationship 
>> between Km value and substrate concentration when making 
>> cocrystallization? How can I know the substrate is enough
for binding?
>> Thank you very much!
>> liuqing
>>
>>

>> 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 
>> <http://im.live.cn/newsexpress>
>
>
>-- 
>Dr. Jeroen R. Mesters
>Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
>Institut für Biochemie, Universität zu Lübeck
>Zentrum für Medizinische Struktur- und Zellbiologie
>Ratzeburger Allee 160, D-23538 Lübeck
>Tel: +49-451-5004065, Fax: +49-451-5004068
>Http://www.biochem.uni-luebeck.de
>Http://www.iobcr.org
>Http://www.selfish-brain.org
>Http://www.opticryst.org
>--
>If you can look into the seeds of time and say
>which grain will grow and which will not - speak then to me 
(Macbeth)
>--
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them 
  both in one book
Please do take a 
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] Fwd: Re: [ccp4bb] co-crystallization

[Roger Rowlett]

  
  
Successful complexation depends on the
  concentration of protein, ligand, and the Kd of the protein-ligand
  complex. For Kd>>[protein], you will probably require
[ligand] > 10 x Kd. As Kd approaches [protein], slightly
superstoichiometric quantities will be sufficient for full
occupancy. For Kd < [protein], stoichiometric quantities of
ligand will suffice. Basically you need a [ligand] that puts near
saturation on the binding isotherm.

Cheers.

On 8/25/2011 2:03 AM, Yvonne TAN Yih Wan wrote:

  
  
  
  

  Hi

  ,
   
  I
  am co-crystallizing a protein with compound and would like
  to know how much of compound to add to protein solution to
  start with. I know that the protein binds compound in a 1
  to 1 ratio but also noticed that the compound precipitates
  out of solution when DMSO is diluted off. Where should I
  start of? A 1 protein :2 compound ratio or more? And what
  is the best method to determine if the binding is
  homogeneous (that all protein has got a compound in it)?
   
  Any

  suggestions would help. Thanks
   
  TY

  
  -- 

   Roger S. Rowlett
  Gordon & Dorothy Kline Professor
  Department of Chemistry
  Colgate University
  13 Oak Drive
  Hamilton, NY 13346
  
  tel: (315)-228-7245
  ofc: (315)-228-7395
  fax: (315)-228-7935
  email: rrowl...@colgate.edu
 

  

[ccp4bb] co-crystallization VS crystal soaking

[rainfieldcn]

Hi, friends:
Is there any published paper describing the case study of the difference 
between co-crystallization and crystal soaking?
I mean has anybody observed different structures by these two methods?
Thank you!

Rain Fieldcn
2010-05-15

[ccp4bb] co-crystallization VS crystal soaking

Hi, friends:
Is there any published paper describing the case study of the difference 
between co-crystallization and crystal soaking?
I mean has anybody observed different structures by these two methods?
Thank you!

[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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Re: [ccp4bb] Fwd: Re: [ccp4bb] co-crystallization

[Herman . Schreuder]

If you do the calculations, you will find that you need a FREE ligand 
concentration of >10 * Kd to get >90% occupancy of the binding site.
If you have e.g. a ligand with a Kd of 100 nM, you would need a free ligand 
concentration of 1 µM. However, a solution of 10 mg/ml of a protein of 30 kDa, 
has a protein concentration of 333 µM, so in theory you should have a total 
ligand concentration (free + bound) of 334 µM. In pratice, some of the ligand 
may have been degraded during (prolonged) storaged, or the compound may not be 
as pure as the chemist would have wished it to be, so it is wise to use a 
safety margin of at least two. We normally use 1-2 mM compound to be on the 
safe side. Having too much ligand usually does not hurt, except that you use 
more compound, but with too little ligand you end up with an empty binding site 
and you will have to repeat the experiment with more ligand.
 
Best,
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Roger Rowlett
Sent: Thursday, August 25, 2011 6:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fwd: Re: [ccp4bb] co-crystallization


Successful complexation depends on the concentration of protein, 
ligand, and the Kd of the protein-ligand complex. For Kd>>[protein], you will 
probably require [ligand] > 10 x Kd. As Kd approaches [protein], slightly 
superstoichiometric quantities will be sufficient for full occupancy. For Kd < 
[protein], stoichiometric quantities of ligand will suffice. Basically you need 
a [ligand] that puts near saturation on the binding isotherm.

Cheers.

On 8/25/2011 2:03 AM, Yvonne TAN Yih Wan wrote: 

Hi ,



I am co-crystallizing a protein with compound and would like to 
know how much of compound to add to protein solution to start with. I know that 
the protein binds compound in a 1 to 1 ratio but also noticed that the compound 
precipitates out of solution when DMSO is diluted off. Where should I start of? 
A 1 protein :2 compound ratio or more? And what is the best method to determine 
if the binding is homogeneous (that all protein has got a compound in it)?



Any suggestions would help. Thanks



TY

-- 



Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

Re: [ccp4bb] co-crystallization VS crystal soaking

[Jürgen Bosch]

Hi Rain,

try both is my advice.
In some cases we (www.sgpp.org) were unsuccessful in soaking but successful in 
co-crystallization. I assume your question is directed towards ligands. If you 
are in the comfortable situation of having your own structure at hand, check 
out a) crystal lattice contacts - would they hamper soaking by restricting 
access to your site ? b) do you want to exchange an existing ligand/co-factor 
in your protein, then it's probably more likely to occur in solution c) how's 
your ligand behaving under your crystallization condition, if it crashes out 
try to form a complex in solution and then co-crystallize.
Another tip, use your apo-form crystals to streak seed crystallization attempts 
with new ligands. When you co-crystallize play with the molarity ratio of your 
ligand.

Jürgen

Bosch et al. Using fragment cocktail crystallography to assist inhibitor design 
of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem (2006) 
vol. 49 (20) pp. 5939-46

@Eric, can you comment on this:
Ojo et al. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for 
selective kinase inhibitors. Nat Struct Mol Biol (2010) vol. 17 (5) pp. 602-7


On May 15, 2010, at 7:47 AM, rainfieldcn wrote:

> Hi, friends:
> Is there any published paper describing the case study of the difference 
> between co-crystallization and crystal soaking?
> I mean has anybody observed different structures by these two methods?
> Thank you!
>   
> Rain Fieldcn
> 2010-05-15

-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] co-crystallization VS crystal soaking

[Bernhard Rupp]

Quite useful ref:

 

1.Danley D (2006) Crystallization to obtain protein-ligand complexes for
structure-aided drug design. Acta Crystallogr. D62(Pt 6), 569-575.

 

http://scripts.iucr.org/cgi-bin/paper?S0907444906012601

 

BR

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jürgen
Bosch
Sent: Saturday, May 15, 2010 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] co-crystallization VS crystal soaking

 

Hi Rain,

 

try both is my advice.

In some cases we (www.sgpp.org) were unsuccessful in soaking but successful
in co-crystallization. I assume your question is directed towards ligands.
If you are in the comfortable situation of having your own structure at
hand, check out a) crystal lattice contacts - would they hamper soaking by
restricting access to your site ? b) do you want to exchange an existing
ligand/co-factor in your protein, then it's probably more likely to occur in
solution c) how's your ligand behaving under your crystallization condition,
if it crashes out try to form a complex in solution and then co-crystallize.

Another tip, use your apo-form crystals to streak seed crystallization
attempts with new ligands. When you co-crystallize play with the molarity
ratio of your ligand.

 

Jürgen

 

Bosch et al. Using fragment cocktail crystallography to assist inhibitor
design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med
Chem (2006) vol. 49 (20) pp. 5939-46

 

@Eric, can you comment on this:

Ojo et al. Toxoplasma gondii calcium-dependent protein kinase 1 is a target
for selective kinase inhibitors. Nat Struct Mol Biol (2010) vol. 17 (5) pp.
602-7

 

 

On May 15, 2010, at 7:47 AM, rainfieldcn wrote:





Hi, friends:
Is there any published paper describing the case study of the difference
between co-crystallization and crystal soaking?
I mean has anybody observed different structures by these two methods?
Thank you!

Rain Fieldcn
2010-05-15

 

-

Jürgen Bosch

Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/> 

 

Re: [ccp4bb] co-crystallization VS crystal soaking

Thank you for your valuable advice.
My situation is this:
I got two completely different structures by soaking and co-crystallization.
I believe co-crystallization. But I don't know if I can find the literature 
which I would cite as a proof.

Rain Field

Re: [ccp4bb] co-crystallization VS crystal soaking

[Anastassis Perrakis]

Hi -

You should not use literature as proof; every protein is different.
You would need to do site directed mutagenesis experiments,
to show which binding mode is relevant.

I am also not sure what 'completely different' means, if the ligands  
are in different sites,

it could also be of interest, eg an allosteric effect.

A.

On 17 May 2010, at 4:20,   wrote:


Thank you for your valuable advice.
My situation is this:
I got two completely different structures by soaking and co- 
crystallization.
I believe co-crystallization. But I don't know if I can find the  
literature which I would cite as a proof.


Rain Field

Re: [ccp4bb] co-crystallization VS crystal soaking

My Friends:
Thank you all, I'll consider your advice!
Best Wishes!
Rain Field

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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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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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

[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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[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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**



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