Bernhard, 

I'm often amazed at how forgiving protein crystallization is, or to put it another way, how efficient screens are at picking up crystallization hits even when you do everything wrong.

However if you do go down to 20 + 20 nl drops you have to remember that probably 3/4 of your protein will be lost by sticking to the plastic of the plate or forming a skin at the air interface.

You generally get roughly the same number of hits from 1 + 1 ul drops (where most of the protein is still in solution) as from 100 + 100 nl drops (roughly half the protein is lost), although the hits will be in different places.  This implies that, within broad limits, things like protein concentration don't matter too much.

My view of crystallization is that there's a group of proteins (lysozyme and many others) where all you have to do is gently push the protein out of solution, while taking care of nucleation (think microseeding).  For these proteins it's a matter of not adding something that interferes with crystallization.  Then there are others where you have to add something to the mix that stabilizes the crystal lattice.  A smaller group requires 2 additives, a few may need 3 ....  At the end there's a bunch that will never crystallize no matter what you do.

It seems that protein concentration is of secondary importance in screening - although I accept that it may be crucial in optimization.

Patrick



On 11 June 2013 08:48, Bernhard Rupp <[log in to unmask]> wrote:

Hmmmm….the real message from figure 3.  The protein concentration…

seems to be that >70% of proteins do NOT crystallize in the 10-12.5 mg bin,

i.e. the ‘right’ concentration is an individual protein-in-that-buffer property - and

all one can say is that the concentration needs to be high enough so that the crystallization

conditions (either right away in case of batch, or vapor-diffusion assisted) can drive

the system across the solubility limit into supersaturation.

Most people actually working with their protein have already a reasonably developed idea what

their protein can take in various buffer systems (valuable parameter!)

… once they have scraped if off a centricon membrane, for example…

The pre-screening originated from structural genomics when one had no idea what

the ‘customer’ actually sent to the facility. If one knows the material it might be more

efficient (i.e. more information from the same precious material) to set up a plate of

96 20+20 nl nanodrops than spending 2 ul on prescreening.

 

Just an opinion sans statistics, BR

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Debasish Chattopadhyay
Sent: Tuesday, June 11, 2013 1:02 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Protein concentration for crystallization

 

Perhaps my question was not expressed well.  I wanted to know if proteins crystallize more frequently when the protein concentration is in the range 5-30mg/ml.

The answer pointed out by my colleague Todd Green is on the page

http://www.douglas.co.uk/PDB_data.htm

 

Thanks for your inputs.

 

Debasish

 

From: Orru, Roberto [mailto:[log in to unmask]]
Sent: Monday, June 10, 2013 5:04 PM
To: Debasish Chattopadhyay
Subject: RE: Protein concentration for crystallization

 

Dear Debasish,

On my memory there are 2 way (but I cannot say that are the only 2!)
First: if you have the structure and you know the water content, you can guess the amount of protein crystallized in your drop by calculating the volume of the crystals.
Second (if you can waste your drops): Fish all the crytsals in any drop for a given concentration, load a sds page w/ silver staining developing and compare it with a calibration curve done with your same protein in the same gel.

Best
R.

From: CCP4 bulletin board [[log in to unmask]] on behalf of Debasish Chattopadhyay [[log in to unmask]]
Sent: Monday, June 10, 2013 10:49
To: [log in to unmask]
Subject: [ccp4bb] Protein concentration for crystallization

What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg?

 

Debasish Chattopadhyay

 

University of Alabama at Birmingham

CBSE-250

1025 18th Street South, Birmingham, Al-35294

USA

Ph: (205)934-0124; Fax: (205)934-0480

 

 


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