It makes slight sense if the word 'respect' can be correlated with whether the drop is mixed (protein+ppt). i have heard some protein may indeed crystallize if it were not mixed or vice-versa (do not expect it with lysozyme though). it would make sense if diffusion played a role but with few ul drop (the tip of pipette covering the surface of the drop in 96-well), i imagine what would be the real reason/advantage for this argument of mixing vs not mixing drops.

--
Karthik

On Mon, Dec 21, 2009 at 4:19 PM, Felix Frolow <[log in to unmask]> wrote:
If "respect" is concerned, try to stop a blow of a sharp samurai sword with  a sharp word.
I myself have no problems whatsoever with the Rigaku recipes. And yes, I have stopped,
at leas once, a blow of samurai sword  with my bare hands. Since than I paint pictures with my left foot.
BTW
In ALL my crystallization attempts I ALWAYS use double distilled (rectified) water. However there is, I must admit, a fume
of triple distilled Jameson around (almost always).  All this is not written in the Rigaku recipe but is very important.

Dr  Felix Frolow

Professor of Structural Biology and Biotechnology

Department of Molecular Microbiology

and Biotechnology

Tel Aviv University 69978, Israel


Acta Crystallographica D, co-editor


e-mail: [log in to unmask]

Tel:           ++972 3640 8723

Fax:          ++972 3640 9407

Cellular:   ++972 547 459 608

On Dec 21, 2009, at 23:02 , [log in to unmask] wrote:

I was going to comment that I have learned the following: "respect" does not mean the same thing in all places in the world. Some time back I had a protein here that I thought needed extra respect and I had learned from a Rigaku employee how to do this - I bowed very very deeply in front of the protein before handling it. But it still did not crystallize. So when I complained to the Rigaku employee about the "recipe", he asked with appropriate hesitation in his voice: are you saying that you only bowed ONCE?

In defense of the original poster, I think the recipe on the Rigaku web site is entirely correct, but it does not specify how to pay proper respect. This was self-evident to the person who wrote down the recipe, but as we all know, what it obvious to one person, is not obvious to the next - especially in such difficult things like respect. Good recipes are indispensable and should be explicit about such important ingredients.

Mark


-----Original Message-----
From: Mark J. van Raaij <[log in to unmask]>
To: [log in to unmask]
Sent: Mon, Dec 21, 2009 12:18 pm
Subject: Re: [ccp4bb] where I have been going wrong in crystallization?

- I think the original poster was only calling attention to the fact that some proteins want to be treated respectfully in order to crystallise (and the fact that Rigaku Japan realises this). I find that indeed the case. 
Other proteins, however, prefer the attitude "I don't know why I am setting up these drops, this protein is too crappy to crystallise", i.e. a challenge. 
Lysozyme, on the other hand, even crystallises under conditions of complete indifference. At least I find that every student in a practical course can get nice crystals of lysozyme, and a majority of these drops have been set up under conditions of complete indifference...maybe lysozyme is not a protein after all, but a salt: lysozyme-chloride / LyCl7 ? 
Mark 
PS the detailed protocols and experiences are useful though. 
 
Quoting Jeffrey Wilson <[log in to unmask]>: 
 
> I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M 
> NaCl method mentioned in a Hampton Research catalog and attributed to 
> Enrico Stura. I see that he has also just commented on this thread. I 
> found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant 
> ratio, lysozyme crystallized in about 1 hour. Jumping that up to 
> 150mg/ml allowed for crystallization in minutes. Hanging drop behaved 
> similarly. I was using lysozyme from Sigma. 
> 
> Jeff 
> 
> Jeffrey Wilson, Ph.D. 
> University of Cincinnati College of Medicine 
> Molecular Genetics Department 
> 231 Albert Sabin Way 
> MSB 3109A 
> Cincinnati, OH 45267-0524 
> (513) 558-1360 
> 
> 
> On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: 
> 
>> Dear All 
>> checking out the Lysozyme crystallization methods on the web I >> liked the Rigaku Instructions that I found: 
>> (http://www.rigaku.com/protein/crystallization.html) 
>> 
>> "...create a drop of 3ul lysozyme solution, and 3 ul of well >> solution, respectfully, for a total drop size of 6ul..." 
>> 
>> So perhaps sometimes I am just not respectful enough to deserve crystals ? 
>> 
>> good wishes to all 
>> regards, 
>> Martyn 
>> ----------------------- 
>> Martyn Symmons 
>> MRC-MBU Cambridge UK 
>> 'Chan fhiosrach mur feòraich.' 
>> Gaelic proverb - 
>> Nothing asked, nothing learned.