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User Guide Overview

Welcome to the py2rely user guide! This section provides comprehensive documentation for all py2rely workflows and features.

Workflows

py2rely is designed around a two-stage strategy:

  1. Screen fast with 2D classification to validate and isolate high-quality particles
  2. Refine thoroughly with 3D sub-tomogram averaging for high-resolution structures

You can use either workflow independently, but combining them gives the best results.

🧪 2D Slab Classification - Fast Particle Screening

Rapidly validate particle picks and isolate coordinates for your protein of interest:

  • Extract thin slabs from tomograms (avoids missing wedge artifacts)
  • Run 2D classification in minutes to hours (vs. days for 3D)
  • Visually inspect class averages to identify your target protein
  • Select high-confidence particles for downstream processing
  • Export cleaned coordinates back to Copick or STAR files

Typical use cases:

  • ✅ Validate a new picking algorithm or template
  • ✅ Separate your protein from contaminants
  • ✅ Identify different conformational states
  • ✅ Quality control before investing in 3D refinement

🔬 3D Sub-tomogram Averaging - High-Resolution 3D Refinement

Process validated particles through the complete sub-tomogram averaging pipeline:

  • Initial model generation (de novo, reference-based, or reconstruction)
  • CTF refinement and Bayesian polishing
  • Post-processing and resolution estimation

Typical use cases:

  • ✅ Determine high-resolution structures
  • ✅ Generate publication-quality maps
  • ✅ Refine particle orientations and CTF parameters

🖥️ Running on HPC with Submitit - Cluster execution with Submitit

Run the STA pipeline on a SLURM cluster with --submitit True so each Relion step is submitted and waited on automatically. Configure Python and Relion load commands with py2rely config; optional GPU/CPU and timeout options are documented in the guide.

For new or uncertain particle picks:

Start with 2D classification to validate/clean particles, then run 3D STA on selected high-quality particles.

For pre-validated particles:

Go directly to 3D sub-tomogram averaging.

Next Steps

  1. Import Data: See Importing Data for data preparation
  2. Screening with 2D Classification: Check out 2D Slab Classification
  3. 3D STA: Read the 3D Sub-tomogram Averaging guide

New to py2rely? Start with the Quick Start Guide for hands-on examples.