[3dem] Lack of Lateral Views

[Brian Gibbons]

Dear Veer,

You can try coating grids with polylysine (or peptides, fibronectin, etc.)
or something that either repels the ring faces, or attracts the ring sides.

You can try adding glucose/glycerol/PEG to the stain solution which can
sometime increase random adsorption to the grid.  This has worked for some,
although this has caused more "fuzzy" appearance when I've done it.

Random conical tilt (RCT) will only work if your particles/rings are NOT
symmetrical so you can align the un-tilted particles before doing the
reconstructions; but tilting or tomography might still give some 3D info.

Best wishes,
Brian








Brian Gibbons, Ph.D.
Stanford University School of Medicine
Structural Biology Department
299 Campus Drive West
Stanford, CA 94305
bgibbons at stanford.edu



On Fri, Oct 5, 2012 at 10:34 AM, Reinhard Rachel <
Reinhard.Rachel at biologie.uni-regensburg.de> wrote:

> >>> 05.10.2012 um 19:15:
> > I am working on a multimeric membrane protein that has a strong
> > tendency to form rings. The top views of these rings can be visualized
> > rather easily using negative-staining EM. However, I am unable to find
> > ANY lateral views. Could someone suggest a solution? CryoEM is not
> > feasible for me at the moment.
> > I use Uranyl Acetate for staining over '300 mesh' carbon (pre)coated
> > copper grids.
>
> dear Veer,
> different treatments of the carbon film ("hydrophobic", i.e. ageing and no
> hydrophilization; vs. hydrophilization = glow discharge with 'no' additive;
> glow discharge with pentylamine; Aebi+Pollard 1987; Dubochet ...). Other
> stains. Combination of these "tricks". Changing the pH of the solution of
> the protein (! 4,5,6,7,8,9 ...) may alter the aggregation state and/or the
> adsorption.
> In fact, you also get some kind of 3D info from negatively stained samples
> by collecting 3D information using the random conical tilting (check
> M.Radermacher's papers), and/or by tomography. Yes, it is limited due to
> staining and air-drying, but it is more than the projection, only.
> kind regards,
> Reinhard
>
> --
> Prof. Dr. Reinhard Rachel
> University of Regensburg
> Centre for EM / Anatomy
> Faculty of Biology & Preclin. Med.
> Universitaetsstrasse 31
> D-93053 Regensburg - Germany
> tel +49 941 943 2837, 1720
> fax +49 941 943 2868
> mail reinhard.rachel at biologie.uni-regensburg.de
> office: VKL 3.1.29
>
> next microscopy conferences:
> http://www.mc2013.de/
> MC2013 in Regensburg, Germany
> http://www.imc2014.com/
> 18th IMC 2014 in Prague, Czech Rep.
>
>
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