Largest cryo-neg stain differences?

[Sacha De Carlo]

Why you guys do not consider to use cryo-negative staining ?

I am not in agreement with what is said below (message from P. Koeck). 
 From my experience with frozen-hydrated cryo-negatively stained samples 
the amplitude contrast contribution is of circa 13%, not 20 nor 25% as 
expected for air-dried negatively stained samples!!

Concerning the flattening, it is not true that projections are not 
affected! Many examples exist showing specimen flattening in 
conventional negative staining.
This has an important effect in the 3D reconstructiuon, which is the aim 
of image analysis...

We could show that cryo-negative staining preserves the structure with 
full or at least partial hydration in some cases.

If one is interested, see "De Carlo et al. (2002). Journal of Struct. 
Biol 138, 216-226"

Yours sincerely,

Sacha




Le Jeudi 19 septembre 2002, à 10:05 , Philip Koeck a écrit :

> I can't offer a good example but some thoughts.
> I'd be grateful for some feed-back from other list members.
>
> If one can assume that:
>
> all cavities of a protein are accessible to stain,
>
> and the main effect of negative stain specimen preparation is a 
> flattening
> of the specimen on the film without changing dimensions in other 
> directions,
>
> then surface rendered reconstructions should look about the same for
> negative stain and cryo as long as the specimen wasn't tilted for
> image recording, since images are projections of the structure anyway.
> (The projection will look the same whether the specimen is flattened or
> not.)
>
> The biggest difference might come from a wrong estimation of amplitude
> contrast during CTF correction (discussed for instance in the EMAN
> manuals and related publications).
>
> I'm not accounting for any changes in conformational details due to 
> drying.
>
>
> Philip Koeck
> Svdertvrns Hvgskola and
> Karolinska Institutet
> Dept. of Bioscience at Novum
> S-14157 Huddinge
> Sweden
> phone: +46-8-6089186
> fax: +46-8-6089290
> http://www.biosci.ki.se/em
>
> _______________________________________
>
>
> ----- Original Message -----
> From: "Ed Gogol" <gogole at umkc.edu>
> To: <3dem at SDSC.EDU>
> Sent: Wednesday, September 18, 2002 8:53 PM
> Subject: Largest cryo-neg stain differences?
>
>
>> I'd like to compare a structural difference we've recently seen 
>> between 3D
>> reconstructions from neg stain and cryEM data with those previously 
>> noted.
>> I can't think of very substantial differences in those structures of 
>> which
>> I am aware, but I'm sure the recipients of this list have some good
>> examples, so I ask you to send me any of which you know (a reference 
>> may
> be
>> helpful, also).
>>
>> Thanks,
>> Ed Gogol
>> Ed Gogol, Associate Professor
>> School of Biological Sciences
>> University of Missouri-Kansas City
>> 5007 Rockhill Rd., Kansas City, MO 64110
>> Ph : (816) 235-2584
>> FAX : (816) 235-1503
>
>
> ----------------------------------------------------
> To unsubscribe from this e-mail list, send mail to
> majordomo at sdsc.edu
> with the words
> unsubscribe 3dem
> in the body of the message.
> Do not send unsubscribe messages to the entire list
> -----------------------------------------------------
>
>
=======================================
De Carlo Sacha
Centre de Microsopie Electronique
27, rue du Bugnon
CH-1005 LAUSANNE
Tél.  0041 21 6925051
URL. http://www.unil.ch/adas/DeCarlo.html
=======================================

----------------------------------------------------
To unsubscribe from this e-mail list, send mail to
majordomo at sdsc.edu
with the words
unsubscribe 3dem
in the body of the message.
Do not send unsubscribe messages to the entire list
-----------------------------------------------------