[3dem] electron dose & data collection strategy for Titan Krios, 300kV, Falcon back thinned

[Sjors Scheres]

Hi Shabih,

We have had good results on our Krios with a pixel size of 1.3 Angstroms 
(i.e. a nominal magnification of 59,000x), using 1 second exposures with 
a dose of ~25 e-/A^2. That should be a small enough pixel to reach the 
3A-end of your target resolution range. If you're capturing movies, you 
may want to consider that beyond a dose of 10 e-/A^2 there will be 
_very_ little signal left at 3 Angstrom resolution. However, depending 
on the shape of your virus (are there nice features to align on or is it 
a featureless ball?) you may still need the higher doses to also get 
strong enough lower frequencies for accurate alignment.

In many cases, we have found it more efficient (both in microscopy time 
and in computing time) to collect a relatively small number of very good 
pictures (manually deleting bad ones as they are collected, and moving 
to a different area of the grid if a given area gives many bad images) 
over collecting many thousands of pictures of widely varying quality. 
But experiences with this may vary, and it will strongly depend on the 
skills of the microscopist, the automation software used, and the 
quality of the grids. In any case, given the icosahedral symmetry of 
your virus, you will not need many particles: several thousand good ones 
should be enough.

Good luck!
Sjors






On 01/13/2014 03:50 PM, Shabih Shakeel wrote:
>
> Dear All,
>
> I am going to collect single particle data on Titan Krios operated at 
> 300kV with Falcon II (back-thinned) detector. The sample is 
> icosahedral virus of 30nm diameter. Does any one have experience 
> collecting data in these settings?
>
> We are especially looking for comments or advice on electron dosage to 
> use and number of frames to record with an aimed resolution of 3-5Å. 
> This is our one shot at collecting the data for this sample, so we 
> really want it to work.
>
> Of course, any other useful comments, advice, tips would also be 
> appreciated.
>
> Thanks a lot.
>
> Best Regards,
> Shabih Shakeel
>
>
> ---------------------------------------------
> Shabih Shakeel, MSc
> Doctoral researcher
> Butcher group
> Institute of Biotechnology
> P.O. Box 65 (Viikinkaari 1)
> 00014 University of Helsinki
> Finland
>
> cell: +358 46 8430774
>
>
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> 3dem at ncmir.ucsd.edu
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-- 
Sjors Scheres
MRC Laboratory of Molecular Biology
Francis Crick Avenue, Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44 (0)1223 267061
http://www2.mrc-lmb.cam.ac.uk/groups/scheres