Dear Kavya,
with a KD of 50 micro-molar, and a highest ligand concentration of 250 micro-molar, you re just 5 times above the KD. I cannot image that you will have 95% of complex then. Usually you will have 90% binding when 10fold above the KD.
With a KD of 50 micro-molar it is going to be tricky to obtain crystals, but not impossible. Even complexes with Kd> 80-90 micro-molar have crystallised and the structure determined.
Best,
Sebastian
> On 11 Feb 2016, at 08:10, Kavyashree Manjunath <[log in to unmask]> wrote:
>
> Dear George,
>
> For one of the ligand the kd was 50 micromole, the
> protein concentration used in MST was 20nM and at
> 250micromolar ligand concentration, it was ~95% bound.
>
> Thank you
> Regards
> Kavya
>
>
>> Almost means 88%? 93% ? 95%? or 99%?
>>
>> That why it is much easier when Kd is known because we do not need all the
>> above detail information just one number (Kd).
>> Give me also the protein concentration of protein during bioassay.
>>
>> G.
>>
>> -----Original Message-----
>> From: [log in to unmask] [mailto:[log in to unmask]]
>> Sent: Wednesday, February 10, 2016 1:17 PM
>> To: George Kontopidis
>> Cc: [log in to unmask]
>> Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
>>
>> Dear George,
>>
>> Sorry for the late reply. 1:500 the MST curve almost reaches the
>> saturation.
>>
>> Thank you
>> Regards
>> Kavya
>>
>>> Dear Kavya,
>>>
>>> The biophysical techniques like MST shows a binding of 20%, 40% or
>>> 99% (protein:ligand)?
>>> when molar ratio of protein:ligand is greater than 1:500 we need this
>>> number to give you an answer
>>>
>>> All depend of the Ka or Kd (1/Ka). That why you have obtained ligand
>>> bound structure even with molar ratio of protein:ligand down to 1:10.
>>>
>>>
>>> Regards,
>>>
>>> George
>>>
>>> -----Original Message-----
>>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
>>> Kavyashree Manjunath
>>> Sent: Tuesday, February 09, 2016 7:30 AM
>>> To: [log in to unmask]
>>> Subject: [ccp4bb] Protein:ligand ratio for co-crystallization
>>>
>>> Dear users,
>>>
>>> I would like to know the minimum protein:ligand ratio that will yield
>>> a cocrystal, provided there are some binding values from biophysical
>>> techniques.
>>>
>>> Suppose, biophysical techniques like MST shows a binding when molar
>>> ratio of protein:ligand is greater than 1:500, is it necessary to have
>>> this ratio while co-crystallizing?
>>>
>>> I have previous experiences in which I had obtained ligand bound
>>> structure when I used 1:10 to 1:30 (molar ratio). But, it is not working
>> now.
>>>
>>> Thank you
>>> Regards
>>> Kavya
>>>
>>>
>>> --
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>>>
>>
>>
>>
>>
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