Tomography tutorial (EMPIAR-10164)

This tutorial shows how to convert raw tilt-series from EMPIAR-10164 (HIV-1 Gag) into a ~3A resolution structure.

Total running time required to complete this tutorial: ~20 hrs.

We first use the command line to download and decompress a tbz file containing a subset of 5 tilt-series (down-sampled 2x compared to the original data), and an initial model:

# cd to a location in the shared file system and run:

tar xvfz nextpyp_tomo_tutorial.tbz

Open your browser and navigate to the url of your nextPYP instance (e.g.,

Step 1: Create a new project

Step 2: Import raw tilt-series

Step 3: Pre-processing


While on the Tilt Series tab, use the navigation bar at the top of the page to look at the results for other tilt-series

Step 4 (optional): Virion segmentation


Click on > Keyboard shortcuts (under the virion image) to reveal instructions on how to speed up the threshold selection process

Step 5: Particle detection

Step 6: Reference-based refinement

Step 7. Fully constrained refinement


Use the navigation bar at the top left of the page to look at the results for different iterations

Step 8. Filter particles

Step 9 (optional): Permanently remove bad particles

Step 10. Region-based local refinement (before masking)

Step 11: Create shape mask


You may need to adjust the binarization threshold to obtain a mask that includes the protein density and excludes the background (a pre-calculated mask is provided with the raw data if you rather use that).

Step 12: Region-based constrained refinement

Step 13: Particle-based CTF refinement

Step 14: Movie frame refinement

Step 15: Refinement after movie frame refinement

Step 16: Map sharpening


Running times were measured running all tilt-series in parallel on nodes with 124 vCPUs, 720GB RAM, and 3TB of local SSDs