In addition to the TranSPHIRE related questions, we provide an in-depth SPHIRE tutorial on our SPHIRE website: Tutorial. There, not only SPHIRE related topics are discussed, but also general tips and tricks for Single Particle Analysis.

Frames and jpg/meta files are stored in different locations, is that a problem?

No, TranSPHIRE offers the possibility to provide a path to the frames and a path to the jpg/meta data. If you setup the TranSPHIRE session after the first images has been collected, TranSPHIRE will try to search for the respective directories itself.

How can I handle multiple gain references in one session?

TranSPHIRE assigns the provided gain reference while finding new files and assigns the currently used gain with the found image. Therefore, every input image is linked to it’s respective gain reference. To set a new gain reference for new images, press the Stop button, provide the new gain reference, and click the Start button to continue.

What is the output folder structure of TranSPHIRE?

For a detailed explanation about the output folders of TranSPHIRE, please visit TranSPHIRE outputs.

How to create a template for faster setup?

You can find more information here: Basic TranSPHIRE setup.

What kind of computer should I use?

TranSPHIRE is designed to run on a Linux system. The better the hardware is, the faster the processing will be. This also allows to stay on-the-fly for faster data acquisition schemes.

You can find more information here: Hardware recommendations.

How do I measure my particle radius in pixels?

There are mutliple ways of doing this, but we recommend to use e2display.py from the SPHIRE package.

>>> e2display.py example_image.mrc

Press the Middle mouse button and the Meas tab. Keep the A/Pix value to 1.0 and click+drag a line on the micrograph. The Len value shows the distance in pixels.

If the particles do not have a globular shape you should choose a radius that is a wider than the measured one to allow for a more liberal centering.

What box size should I use?

A list of good box sizes can be found on the EMAN2 box size recommendation website.

The recommendation is to use a box size of 1.5x to 2x of the longest particle axis (3x to 4x of the radius).

By default, TranSPHIRE will use the next bigger value of a “good” box size from the list after multiplying the provided protein radius by 3.


Example 1:

particle_radius = 100
box_size = 300 # particle_radius * 3.
final_box_size = 300 # 300 is in the list of good values.

Example 2:

particle_radius = 101
box_size = 303 # parrticle_radius * 3.
final_box_size = 320 # 303 is not in the list of good values, the next larger good value is 320.

How can I see how many particles have been extracted?

To check the number of extracted particles click on

Visualisation -> Plot Extract -> Plot per micrograph -> accepted (lower tab row)

Check the Sum: field of the data statistics area.

How do I cite TranSPHIRE?

To cite TranSPHIRE use the following citation: Stabrin, M., Schoenfeld, F., Wagner, T. et al. TranSPHIRE: automated and feedback-optimized on-the-fly processing for cryo-EM. Nat Commun 11, 5716 (2020). https://doi.org/10.1038/s41467-020-19513-2

Please also properly cite the individual tools that you used during the TranSPHIRE run.