[3dem] Is there an asymmetric biological sample out there that reveals handedness at CET resolution?

[Ariane Briegel]

Dear Christian,
you bring up an important question in tomography. Handedness can change during many steps in data collection and processing, and different software packages follow different conventions.
We have been discussing this issue quite a lot in our lab recently and are actually preparing a manuscript. 
The short answer is to go through data collection and data processing using an asymmetric sample with known handedness. We have been successful using ribosome averaging or T4 phage tails. 
The best test sample, however,  are DNA origami helixes with gold beads attached. You can see the handedness clearly without the need for averaging even at lower magnifications.

Kuzyk, A., Schreiber, R., Fan, Z., Pardatscher, G., Roller, E.-M., Högele, A., Simmel, F. C., et al. (2012). DNA-based self-assembly of chiral plasmonic nanostructures with tailored optical response. Nature, 483(7389), 311–314. doi:10.1038/nature10889

Good luck, 
Martin and Ariane 

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Ariane Briegel, Ph.D.
California Institute of Technology
Broad Center, MC 114-96
1200 E. California Blvd.
Pasadena, CA 91125

Tel: 626-395-8848
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On Aug 7, 2012, at 12:30 PM, Friedrich Foerster wrote:

> hi christian,
> 
> the handedness problem arises because tilt series alignment cannot
> distinguish a tilt axis of x and x+180 degrees. one option is indeed
> to acquire tomograms of something asymmetric like ribosomes.
> alternatively, you do the same thing people do in random conical
> tilting to determine the tilt direction: take micrographs of carbon
> and tilt your sample. the ctf rings right and left of your tilt axis
> will tell you which side is top and which is bottom.
> 
> cheers
> 
> friedrich
> 
> On Tue, Aug 7, 2012 at 8:33 PM, Mike Strauss
> <mikestrauss13 at crystal.harvard.edu> wrote:
>> Hi Christian,
>> 
>> you could use a helical particle and look at the rise and rotation of the subunits.  Perhaps microtubules, or a helical virus.
>> 
>> If you want to see if a sample is loaded in the reverse direction, you could reconstruct a plastic section with fiducially on a known side (i.e. always on top), and then see where they end up in the program.
>> 
>> good luck.
>> 
>> mike
>> 
>> 
>> On Aug 7, 2012, at 2:27 PM, Christian Geiss <geiss at biophysik.org> wrote:
>> 
>>> Hello,
>>> 
>>> I am a Phd student, do cryo electron tomography and have a problem determining the handedness of my sample, because viewers for tomograms like Amira, Matlab-based scripts can invert the handedness depending if you look at your tomogram from top to bottom or vice versa. Since those programs behave to some extent like black boxes, the easiest would be to have a nice asymmetric biological sample where you know the handedness and that the latter can be judged quite easily from a reconstruction, meaning without further processing like subtomogram averaging.
>>> 
>>> So far, I can only think of bacterial ribosomes since they are asymmetric and quite big, but for cryoET the resolution would be not reasonable to resolve the handedness at all. Thus I could need in the ideal case e.g. something like a large complex that links each other to repetitive units with a known handedness.
>>> 
>>> I know its a tough question, but maybe someone can give creative input here? I would highly appreciate it!
>>> 
>>> Best regards,
>>> 
>>> C. Geiss
>>> 
>>> 
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>> 
>> Mike Strauss
>> Harvard Medical School - Dept. BCMP
>> 240 Longwood Ave. - Bldg C2 Rm 125A
>> 02115 Boston, MA, USA
>> (617) 432 1216
>> mikestrauss13 at crystal.harvard.edu
>> 
>> 
>> 
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> 
> 
> 
> -- 
> Dr. Friedrich Foerster
> Max-Planck Institut fuer Biochemie
> Am Klopferspitz 18
> D-82152 Martinsried
> 
> Tel: +49 89 8578 2632
> Fax: +49 89 8578 2641
> 
> www.biochem.mpg.de/foerster
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