[3dem] Cryo-EM size limit

[Sacha De Carlo]

Your "particles" from Fig. 5 are almost as big as your scale bar. From the legend one can read it's corresponding to 200A, or 20nm. That's huge for a 56kDa protein!!!

Euk. RNA polymerase II is about 16nm in its "largest" extension, and it's a 514kDa protein.

Please refrain from using preposterous statements such as "it should be no problem to get an intermediate resolution reconstruction on protein that is less than 100kDa by single-particle cryo-EM".

Any object that is below 200 kDa and more or less globular in shape will be a "tricky" one in terms of obtaining very good alignment parameters (from unstained, frozen-hydrated samples) leading to a 3D map showing 15A details...

Thank you, and a good night,

sacha

================================================
Sacha De Carlo, Assistant Professor
Chemistry Department, Marshak Science Building &
New York Structural Biology Center
The City College of New York
Convent Ave & 138th Street
New York, NY 10031
(212) 650-6070
e-mail: sdecarlo at ccny.cuny.edu
URL: http://www.planetesacha.com
================================================

--- On Wed, 10/27/10, Gang (Gary) Ren <gren at lbl.gov> wrote:

From: Gang (Gary) Ren <gren at lbl.gov>
Subject: RE: [3dem] Cryo-EM size limit
To: "'Reza Khayat'" <rkhayat at scripps.edu>, 3dem at ucsd.edu
Date: Wednesday, October 27, 2010, 10:10 PM

Hi, Reza,
It should be no problem to get an intermediate resolution reconstruction on
protein that is less than 100kDa by single-particle cryo-EM, even by
cryo-electron tomography. An example is that the 56kDa protein (2 copies of
apoA-I, each is 28kDa) in high-density lipoprotein (HDL) can be clearly
visualized by electron cryo-tomography, even using non-FEG microscope. For
details, see Fig 5 in following paper
http://www.jbc.org/content/early/2010/10/25/jbc.M110.187799.full.pdf+html
It is really depended on how you handle your sample preparation, cryo-EM
operation and 3D reconstruction procedure. 

Best wishes,
Gary
 
----------------
Gang (Gary) Ren, PhD
Staff Scientist
Ph: (510) 495-2375; Fx: (510) 486-7268
Email: gren at lbl.gov; Web:http://foundry.lbl.gov/
Lawrence Berkeley National Laboratory
Molecular Foundry, Rm 2220
1 Cyclotron Road, MS 67R2206
Berkeley CA 94720-8197
 

-----Original Message-----
From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On
Behalf Of Reza Khayat
Sent: Wednesday, October 27, 2010 12:34 PM
To: 3dem at ucsd.edu
Subject: [3dem] Cryo-EM size limit

Hi,
    I was wondering if anyone had an opinion on what the smallest  
macromolecular sample (in size or mass) could be for successful  
single particle cryo-EM and image reconstruction?  I'm hoping for a  
resolution better than 15Å.  This would be without the use of phase  
plate technology.

Thanks,
Reza

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