Re: [ccp4bb] co-crystallization

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: CCP4 Study Weekend, SR User Group Meeting programme
From: "Flaig, R (Ralf)" ralf {- dot -} flaig {- at -} DIAMOND {- dot -} AC {- dot -} UK
Date: 2008-12-01

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Subject: Re: Program to fill unitcell randomly
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2008-12-01

Subject: Re: co-crystallization
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2008-12-01

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
>>
>> ------------------------------------------------------------------------
>> å…«å¦å¨±ä¹åŒ…æ‰“å¬ï¼ŒMSNèµ„è®¯é€Ÿé€’å¸®ä½ å¿™ï¼ äº†è§£è¯¦ç»†ï¼
>>
>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: CCP4 Study Weekend, SR User Group Meeting programme
From: "Flaig, R (Ralf)" ralf {- dot -} flaig {- at -} DIAMOND {- dot -} AC {- dot -} UK
Date: 2008-12-01

Next message:
Subject: Re: Program to fill unitcell randomly
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2008-12-01