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

[R.I.Koning at lumc.nl]

Hi Daniel,

The amorphous structures you observed I have seen in pure lipid vesicle suspensions and are top (round disks) and side views (double lines) of flat lipid disks. I cannot exclude that in your case these flattened disks are flattened virus capsids. In that case it might be difficult to remove them from your suspension.

As suggested earlier, the addition of small molecules might greatly alter the adherence of particles to your grid. Alternatively you could think about adding a very thin carbon film to the holey carbon grids (by picking up a thin layer from water) so the viruses also attach in the ‘holes’. Another alternative is to use lacey carbon films with have very little carbon.

Good luck,

Roman

Dr. R.I.Koning

Leiden University Medical Center, Department of Cell Biology, Section Electron Microscopy
Postal Zone S1-P, P.O.Box 9600, 2300 RC, Leiden, The Netherlands

visiting address. Einthovenweg 20, Building 2, zone S1-P, room R-90-20
tel. (31) 71 526 9296 fax. (+31) 71 526 8270 mail. r.i.koning at lumc.nl web. electronmicroscopy.lumc.nl

From: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of LUQUE BUZO, DANIEL
Sent: maandag 20 augustus 2012 15:43
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Virus particles excluded from CryoEM holey grids holes


 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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