[3dem] Cryo-EM size limit

[Michael Landsberg]

Dear Reza,

As Sacha has correctly pointed out, Gary's response is misleading and does not really answer your question principally because:

* The cited example images are claimed to be of a 56kda dimer attached to a presumably much larger HDL particle. This is a very different proposition to analyzing a 56kda particle in isolation.

* The cited example is (as stated in the figure legend) negatively stained, not cryo as you asked.

So to answer your question, my own experience is that cryoEM SPA becomes challenging at 200-300kda and below. Others may share differing opinions though and in my opinion this is because ultimately it comes down to whether you can confidently identify and acquire a homogeneous distribution of your target particle.

Regards
Michael

--
Dr Michael Landsberg
Senior Research Officer
Division of Chemistry & Structural Biology
Institute for Molecular Bioscience
The University of Queensland, St Lucia, QLD 4072, Australia

Email: m.landsberg at uq.edu.au
Phone: +61-7-3346 2010


On 28/10/2010, at 3:05 PM, Sacha De Carlo <sachadecarlo at yahoo.com> wrote:

> 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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