Re: [ccp4bb] control of nucleation

Thu, 06 May 2010 03:13:12 -0700

Dear Zq & CCP4BB readers,

The precipitant is the main component that affects nucleation.

In specific cases other factors can be used to modulate nucleation as mentioned before by others:
protein concentration, temperature, drop size, initial protein/precipitant ratio etc.
All good components of a very long list that will give a student years of work ahead.

Just to take the first item: "Protein concentration"
Most will think that "Protein concentration" should be reduced to reduce nucleation. Unfortunately,
what will happen when the protein concentration is reduced is not so easily predictable.
Let's do the opposite! Just for fun, let's increase the protein concentration instead.
We will increase the protein concentration while severely reducing the precipitant concentration.
The 15 mins lysozyme crystallization is a good example of this.
Lysozyme concentrations of 100-150 mg/ml are used. The high protein concentration allows crystals to grow rapidly, so each nucleus has a chance to grow before more nuclei are formed. This is because each growing nucleus ends up depleting its local environment and making the nucleation of others nearby less likely.
Nucleation requires a higher degree of supersaturation than crystal growth. Getting it to work and controlling it requires accuracy, but it is great fun to do ... and lysozyme is cheap.
HOT STUFF crystallization is ! In the case of lysozyme: Do it in a hot room you for better control. 

This ambiguity persists for other items:
Thomas Edwards suggests that: "dioxane - it is supposed to reduce nucleation."
"Supposed to" when it does not do the opposite:
Ménétrey, J., Perderiset, M., Cicolari, J., Houdusse, A. & Stura, E.A. (2007) Improving Diffraction from 3 to 2 Å for a Complex between a Small GTPase and Its Effector by Analysis of Crystal Contacts and Use of Reverse Screening. Cryst. Growth Des. 7:2140-2146.
This is the one additive that really increases nucleation in the above paper.
Online access:  Improving Diffraction

The procedures in "reverse screening" are used to identify what each proposed effector really does and use it to
achieve better crystals.

As screening is done with smaller and smaller drops, the conditions that will emerge more often are those where the nucleation rate is very high. Nanodrops will yield "overnucleation".

To conclude dear Zq, listen to all the advice, but unless you really understand your protein you will
find that you will achieve the opposite of what you are trying to do.

Enrico.



On Thu, 06 May 2010 10:10:37 +0200, Thomas Edwards <[email protected]> wrote:

> Dear Zq,
>
> A few ideas:
>
> 1) Vary protein concentration, temperature, or protein : mother liquor
> ratio.
> 2) Try dioxane - it is supposed to reduce nucleation.
> 3) give your protein a good hard spin before you set up drops to remove
> aggregates.
> 4) seeded factorial screen.
> 5) re-purify on gel filtration?
>
> Ed
>
> ______________
> T.Edwards Ph.D.
> Garstang 8.53d
> Astbury Centre for Structural Molecular Biology
> University of Leeds, Leeds, LS2 9JT
> Telephone: 0113 343 3031
> http://www.bmb.leeds.ac.uk/staff/tae/
> -- Nature composes some of her loveliest poems for the microscope and
> the telescope. ~Theodore Roszak
>
>
>
> ________________________________
> From: zq deng <[email protected]>
> Reply-To: zq deng <[email protected]>
> Date: Thu, 6 May 2010 09:03:46 +0100
> To: <[email protected]>
> Subject: [ccp4bb] control of nucleation
>
> hello,everybody . due to excess nucleation,I often get many tiny
> crystals instead of few,large crystals.i wana optimize the condition,
> does anyone have adivce about this?
>
> Best regards.


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