UCSF/IND Titan Krios 2 pixel size & sign-up guidelines

 

 

Nominal Magnification

Pixel Size (Å/pix)

Super Resolution

Pixel Size (Å/pix)

Counting mode (or after 2x FT bin)

Dose Rate 

(e-/pix/s); 

Total Dose (e-/A2)

Exposure time (s)

Dose (e-/A2/frame)

120 frames 

Magnification of 5 μm physical pixel (at super res)

64,000 (CDS)

0.673 1.346

8.0 ; 66

15.6

0.55 (0.13s frame)

36284
105,000 (CDS) 0.417 0.834 16.0 ; 46 2.024 0.57 (0.025s frame) 117232

130,000 (CDS)

0.326

0.652

8.0 ; 66

4

0.53 (0.033s frame)

148764

33,000 (CDS) 1.33 2.66        

 

  • Use ilab to book time for Krios2 usage and grid location/details. Please email [email protected] and [email protected] for any further modifications to grid loading and microscope/camera related issues. You can contact any Eric at any time for troubleshooting when/wherever.
  • Magnifications calibrated for image shift vs coma at specified dose rate, exposure time, specific illumination area. If another magnification or dose rate necessary please contact [email protected] to modify.
  • At the beginning of each users session, reference settings files for desired magnification will be loaded and saved onto a ‘n’kx_run_SerialEMsettings.txt file that the user can then modify for their specific image shift, template and paths
  • energy filter tuning will be taken care of weekly, and should not be touched. energy filter zero loss peak is handled in the serialem script but if there are issues with it drifting far off users should make sure that the beam is unblanked and coordinates over an empty hole are updated in the script. If the serialem script isn't working or the zlp is lost, please contact Eric and he will handle it remotely.
  • alignments should not be touched, currently LM mags should be within a few microns with the low dose mags, which also don't need additional beam/image shift or any compensation in serialem. If this is not the case, contact Eric
  • Currently with above settings, T20S proteasome and Apoferritin have been tested on indicated 105kx with image shift reaching 1.9 Å, and 1.5 Å. 105kx and 64kx datasets for aldolase have also been taken with highest resolution datasets at 2.4 Å.
  • 16 eps settings tested for 5x5 image shift (up to ~4-5 μm shift), total exposure and frame exposure adjusted accordingly, test dataset for apoferritin goes to 1.9 Å with standard processing of FTbin 2 data.
  • Pixel sizes have been updated using current benchmark maps for apoferritin by Ming Sun compared to maps generated from PDB 5OBA for 64kx,105kx and 135kx nominal mag

 

Last updated: 07/20/21