[3dem] Virus particles excluded from CryoEM holey grids holes

[Natesh Ramanathan]

Hi Daniel,
         You can try a thin layer of Carbon coating on the C-flat holey
carbon grids.  Typically you can use 1/10th the virion concentration on
this carbon-coated holey grids (CCHG) compared to the concentration that
you would be using for  c-flat holey grids alone.

         However I donot have any suggestion for your question "how to
remove the aggregates (uncharacterized structures) without altering virus
particles".

 Hope that helps.
warm regards
natesh.

-- 
*Dr. Ramanathan Natesh*
Ramalingaswami Fellow-DBT, Assistant Professor
Indian Institute of Science Education and Research, Thiruvananthapuram,
College of Engineering Campus,
Computer Science Building, Kulathur,TVM
695016, Kerala, India

natesh at iisertvm.ac.in
http://www.researcherid.com/rid/C-4488-2008
http://iisertvm.ac.in/~natesh
*Ph. 0091- 471-2599403
Fax.0091-471-2597427*
On 20 August 2012 19:12, LUQUE BUZO, DANIEL <dluque at cnb.csic.es> wrote:

>  Dear all,
> We are having serious problems to obtain high quality cryo-EM holey grids
> of an apparently excellent virus preparation. Find enclosed several cryo-EM
> images taken at different magnifications of the fungal virus preparation we
> are working with. As you can see, virions attach preferentially to the
> carbon surface, whereas holes are almost empty.  We could also observe the
> presence of other larger structures than virions of variable size
> (arrowheads). We suspect that the anomalous behavior of the virus particle
> suspension is due to the presence of these uncharacterized structures.
>
> Particle concentration is routinely tested by negative staining of serial
> dilutions. Although dilution 1/80 is adequate to obtain a reproducible lawn
> of particles by negative staining, we had to use a dilution 1/5 to lay a
> few particles in the holes, ~20-30 particles/hole (as shown in these
> images). We have used many variations in our protocols with negative
> results; we used clean acetone washed grids, with/without glow discharge,
> and Quantifoil and C-flats holey grids with the same results (even using
> undiluted sample). SDS-PAGE and Coomasie staining of the virus preparations
> showed only the viral proteins and no contaminant. We suspect that these
> amorphous structures are related to lipids and/or nucleic acids from the
> virus.
>
> Have anyone had a similar virus sample with this behavior? If so, How can
> we remove these aggregates without altering virus particles? .
>
> You can get the images from this web site:
>
> http://halley.cnb.csic.es/~virus/3dem/CryoEM_problems.jpg
>
> Thanks in advance!
>
> Best regards,
>
> --
>
> Daniel Luque, Ph.D.
> Department of Structure of Macromolecules
> Centro Nacional Biotecnología/CSIC
> Campus de Cantoblanco
> C/ Darwin nº 3.
> 28049 Madrid, Spain
>
>
>
>
>
>
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